Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

  1. Yang Meng
  2. Yue Ni
  3. Zhuoran Li
  4. Tianhang Jiang
  5. Tianshu Sun
  6. Yanjian Li
  7. Xindi Gao
  8. Hailong Li
  9. Chenhao Suo
  10. Chao Li
  11. Sheng Yang
  12. Tian Lan
  13. Guojian Liao
  14. Tongbao Liu
  15. Ping Wang
  16. Chen Ding  Is a corresponding author
  1. College of Life and Health Sciences, Northeastern University, China
  2. Department of Scientific Research, Chinese Academy of Medical Sciences and Peking Union Medical College, China
  3. NHC Key Laboratory of AIDS Immunology, The First Affiliated Hospital of China Medical University, China
  4. College of Pharmaceutical Sciences, Southwest University, China
  5. Medical Research Institute, Southwest University, China
  6. Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center New Orleans, United States

Abstract

Cryptococcus neoformans poses a threat to human health, but anticryptococcal therapy is hampered by the emergence of drug resistance, whose underlying mechanisms remain poorly understood. Herein, we discovered that Isw1, an imitation switch chromatin remodeling ATPase, functions as a master modulator of genes responsible for in vivo and in vitro multidrug resistance in C. neoformans. Cells with the disrupted ISW1 gene exhibited profound resistance to multiple antifungal drugs. Mass spectrometry analysis revealed that Isw1 is both acetylated and ubiquitinated, suggesting that an interplay between these two modification events exists to govern Isw1 function. Mutagenesis studies of acetylation and ubiquitination sites revealed that the acetylation status of Isw1K97 coordinates with its ubiquitination processes at Isw1K113 and Isw1K441 through modulating the interaction between Isw1 and Cdc4, an E3 ligase. Additionally, clinical isolates of C. neoformans overexpressing the degradation-resistant ISW1K97Q allele showed impaired drug-resistant phenotypes. Collectively, our studies revealed a sophisticated acetylation–Isw1–ubiquitination regulation axis that controls multidrug resistance in C. neoformans.

Editor's evaluation

This important study makes a solid connection between chromatin remodeling, post-translational regulation, and antifungal drug resistance in Cryptococcus neoformans, revealing a new facet of how drug resistance can emerge. Establishing a link between chromatin remodeling and antifungal resistance is a finding that will be of interest to infectious disease researchers, cell biologists, and drug developers.

https://doi.org/10.7554/eLife.85728.sa0

Introduction

Emerging and re-emerging fungal pathogens are one of the primary causes of infectious diseases resulting in mortalities in humans and animals (Brown et al., 2012; Fisher et al., 2012; Gnat et al., 2021). It is estimated that approximately 300 million people suffer from fungal-related diseases, subsequently leading to 1.6 million deaths annually (Stop neglecting fungi, 2017). Additionally, fungal infection leads to substantial population declines in animals and amphibians, even threatening their extinction (Fisher and Garner, 2020; Fisher et al., 2012; Scheele et al., 2019). Intriguingly, certain pathogenic fungi are linked with cancer development (Hosseini et al., 2022; Narunsky-Haziza et al., 2022; Saftien et al., 2023). The World Health Organization has recently published the first-ever list of fungi posing threats to human health that lists Cryptococcus neoformans as one of four high-priority infection agents (World Health Organization, 2022). Cryptococcus species, which cause meningoencephalitis and acute pulmonary infections, are responsible for 15% of the global HIV/AIDS-related fatalities (Erwig and Gow, 2016; Kronstad et al., 2012), totaling an estimated 220,000 deaths per year (Iyer et al., 2021; Ngan et al., 2022; Rajasingham et al., 2017; Tugume et al., 2023). Recently, it has been reported that Cryptococcus is also involved in secondary infections in people with COVID-19 (Khatib et al., 2021; Spadari et al., 2020; Woldie et al., 2020).

Cryptococcosis has a 100% death rate if patients are left untreated, but treatment remains challenging because the number of available antifungal medications is limited. Amphotericin B (Amp B) and azoles constitute the primary treatment options, with Amp B often in combination of 5-fluorocytosine (5-FC) (Billmyre et al., 2020; Molloy et al., 2018; Spadari et al., 2020). In light of the treatment limitations and risks, as well as the high costs associated with developing new antifungal treatments, the US FDA has categorized anti-cryptococcal therapies as ‘orphan drugs’, granting regulatory support by reducing the requirements for clinical studies (Denning and Bromley, 2015). Nevertheless, resistance to anti-cryptococcal drugs occurs rapidly, outpacing the development of new therapeutic options.

The mechanisms by which antifungal resistance occurs can be classified as either mutation in drug targets or epigenetic phenomena (Lee et al., 2023; Li et al., 2020; Wan et al., 2021). Point mutations in binding domains or regions of the drug target proteins often hinder their interactions with drugs resulting in resistance (Sionov et al., 2012; Wan et al., 2021), such as in the case of lanosterol demethylase, encoded by the ERG11 gene, in Candida and Cryptococcus species (Bosco-Borgeat et al., 2016; Sionov et al., 2012). Additionally, pathogenic fungi display a disparate set of mutations that counteract various antifungal drugs. Defects in DNA mismatch repairs resulting in high gene mutation rates and, thereby, 5-FC resistance was identified in C. deuterogattii, a species distinct but close to C. neoformans (Billmyre et al., 2020). Mutations in genes encoding the cytosine permease (Fcy2), uracil phosphoribosyl transferase (Fur1), and UDP-glucuronic acid decarboxylase (Uxs1) conferring 5-FC resistance were also identified in multiple clinical cryptococcal isolates (Billmyre et al., 2020; Chang et al., 2021).

In comparison, nondrug target-induced resistance often refers to the association between mutations and altered gene expressions in regulatable components of sterol biosynthesis and efflux drug pumps (Coste et al., 2004; Dunkel et al., 2008; Morschhäuser et al., 2007; Silver et al., 2004). Studies of C. albicans transcription factor Tac1 illustrate that gain-of-function mutations or alterations in gene copy numbers regulate efflux pump genes (Coste et al., 2004). Gain-of-function mutations were also identified in C. albicans transcription factors Upc2 and Mrr1, which activate ERG11 gene expression and drug pumps, respectively (Dunkel et al., 2008; Silver et al., 2004). A Upc2 homolog was identified in C. neoformans to be involved in regulating steroid biosynthesis, but its role in drug resistance was not clear (Kim et al., 2010). Other proteins capable of modulating efflux pump gene transcription in Cryptococcus remain unknown.

Still, a significant number of clinical isolates demonstrated resistance to multiple drugs, despite the absence of alterations in genes associated with drug resistance (Malavia-Jones et al., 2023; Rajasingham et al., 2017; Sun et al., 2014). Recent evidence indicates that epiregulation by protein posttranslational modification (PTM) mediates drug resistance in pathogenic fungi (Calo et al., 2014; Robbins et al., 2012). The acetylation of the heat-shock protein Hsp90 mediates antifungal drug response in Saccharomyces cerevisiae and C. albicans by blocking the interaction between Hsp90 and calcineurin (Robbins et al., 2012). Despite this observation, the functions of substantially acetylated proteins in antifungal drug resistance remain unknown (Li et al., 2019). Other important PTMs, such as ubiquitination, remain uninvestigated in human fungal pathogens.

Chromatin remodeling factors have significant roles in several physiological activities, such as gene transcription in response to environmental stressors. The ISWI (Imitation Switch) family is a well conserved group of chromatin remodeling families. The ISWI has a highly conserved ATPase domain from the SWI2/SNF2 family, which belongs to the superfamily of DEAD/H-helicases. This domain is responsible for driving chromatin remodeling. Additionally, the protein has distinctive HAND-SANT-SLIDE domains that are involved in binding to DNA. The presence of ISWI ATPase in chromatin remodeling complexes, including as NURF, CHRAC, and ACF, was first discovered in Drosophila homologs (Tyagi et al., 2016). Subsequently, it was found that these complexes are widely conserved in various other taxa, including yeast and mammals. ISWI complexes from different taxa have variations in their subunits. In Drosophila, there is a single ISWI gene that forms three separate complexes known as NURF, CHRAC, and ACF (Längst and Becker, 2001). The ISWI–ACF complex in Neurospora crassa is essential for repressing a certain set of genes that have been methylated at the H3K27, as well as genes targeted by PRC2 (polycomb repressed complex 2) (Kamei et al., 2021; Wiles et al., 2022). The baker’s yeast contains two similar versions of Isws, known as Isw1 and Isw2. Isw1 forms associations with either Ioc3 or Ioc2 and Ioc4 to create two separate remodeling complexes, known as Isw1a and Isw1b, respectively (Vary et al., 2003). The S. cerevisiae Isw2 protein has the capability to interact with Itc1, forming a complex that can position the remodeler during the process of nucleosome sliding (Kagalwala et al., 2004). The ISWI mechanism in S. cerevisiae functions with assistance from other modulators. For instance, the Isw1b complex acts in conjugation with Chd1 to regulate chromatin organization (Smolle et al., 2012), and the ability of Isw2 to attach to DNA might be dependent on Sua7 (Yadon et al., 2013).

Nevertheless, there is currently no information available regarding ISWI in C. neoformans. In this study, we identified a conserved chromatin remodeler, Isw1, from C. neoformans, and we demonstrated its critical function in modulating gene expression responsible for multidrug resistance, as the isw1 null mutant is resistant to azoles, 5-FC, and 5-fluorouracil (5-FU). We further demonstrated that Isw1 typifies a PTM interplay between acetylation and ubiquitination in regulating an Isw1 ubiquitin-mediated proteasome axis in response to antifungal exposure. Dissection of PTM sites on Isw1 revealed an essential reciprocal function of Isw1K97 acetylation in modulating Isw1’s binding to an E3 ligase, Cdc4, which initiates a ubiquitination–proteasome degradation process. Finally, we showed that the PTM interplay mechanism occurs horizontally in clinical strains of C. neoformans.

Results

Cryptococcal Isw1 represses drug resistance

Our previous studies demonstrated the indispensable function of acetylation in modulating the pathogenicity of C. neoformans (Li et al., 2019). Through screening our in-house acetylome knockout library, we found that a single homolog of imitation switch (ISWI)-class ATPase subunit, Isw1, is a critical modulator of multidrug resistance in C. neoformans. This is in contrast to S. cerevisiae in which whole-genome duplication led to the presence of two paralogs, Isw1 and Isw2 (Kellis et al., 2004; Tsukiyama et al., 1999; Wolfe and Shields, 1997). We have generated the cryptococcal isw1Δ mutant and complemented the mutant with the wild-type ISW1 tagged with a Flag sequence (Supplementary file 1). The isw1Δ mutant exhibited profound resistance to azole compounds, including fluconazole (FLC), ketoconazole (KTC), 5-FC, and 5-FU, but not to the polyene compound Amp B (Figure 1a), in comparison to the wild-type and the complemented strains. Both liquid growth and agar spotting assays consistently showed robust resistance to all four antifungal compounds by the isw1Δ mutant strain (Figure 1b).

Figure 1 with 1 supplement see all
Isw1 represses the expression of drug-resistance genes.

(a) Spotting assays of ISW1 mutant strains. Wild-type H99, isw1Δ, and ISW1 complementation strains were spotted onto YPD agar supplemented with indicated concentrations of antifungal agents. Plates were incubated at 30°C for 3 days. (b) Drug inhibitory tests. The H99 and isw1Δ (n = 3 each) strains were tested to determine the drug inhibition of several antifungal agents. Twofold dilutions of fluconazole (FLC) from 0 to 64 µg/ml, ketoconazole (KTC) from 0 to 2 µg/ml, or 5-fluorocytosine (5-FC) from 0 to 400 µg/ml were added to YPD medium. After 24 hr at 30°C, absorbance at 600 nm was used to measure growth. The optical densities of duplicate measurements were averaged and normalized relative to the control without FLC. Quantitative data were depicted in color (see color bar) and bar plots. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (c) Transcriptome analysis of isw1Δ. Samples of RNA were isolated from H99 and isw1Δ cells (n = 3 each) supplemented with or without 10 μg/ml FLC. Transcriptome analysis was performed, and a heat map of the expressions of drug-resistance genes was generated. Statistical significant genes are labeled with asterisks. (d) Intracellular concentration of FLC. H99 and isw1Δ cells (n = 3 each) were incubated in the presence of 40 μg/ml FLC at 30°C for 5 hr. Cells were then washed and weighed, and the intracellular FLC was quantified using high-performance liquid chromatography. A two-tailed unpaired t-test was used. Data are expressed as mean ± SD. (e) Scheme of the mechanism for 5-FC and 5-fluorouracil (5-FU) resistance. The red arrow indicates the entry of 5-FC via Fcy2. Green arrows indicate the downregulation of gene expression in response to 5-FC in the isw1Δ strain. (f) Analyses of 5-FC-resistance genes using quantitative reverse transcription PCR(qRT-PCR). Indicated strains (n = 3 each) were grown with or without 400 μg/ml 5-FC, then qRT-PCR was performed to determine gene expressions of FCY1, FUR1, UGD1, UXS1, and FCY2. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD. (g) Chromatin immunoprecipitation (ChIP)-PCR analysis of Isw1-FLAG. ISW1-FLAG cells (n = 3) were incubated without drug treatment at 30°C for 5 hr. ChIP-PCRs were performed in the ISW1-FLAG strain (n = 3 each). Isw1 target genes, INO1 and INO80, were amplified and used as positive controls. CNAG_06338 were used as a negative control. Potential target genes, CNAG_03600, UXS1, UGD1, FCY1, and FCY2 were tested. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD. (h) Truncation analysis of cryptococcal Isw1. Truncated Isw1s were cloned and transformed into the isw1Δ strain to generate the DNA-binding domain (DBD) truncation (isw1Δ/ISW1DBDΔ), SNF truncation (isw1Δ/ISW1SNFΔ) and helicase C truncation (isw1Δ/ISW1Helicase-CΔ) strains, which were spotted onto YPD agar plates supplemented with indicated antifungal drugs. (i) qRT-PCR analysis in truncated strains supplemented with 5-FC or FLC. Indicated strains were treated with 5-FC or FLC. RNA samples were isolated. qRT-PCRs were performed using oligos from UGD1, UXS1, FCY2, CNAG_03600, and CNAG_06338.

To examine whether Isw1-regulated multidrug resistance occurs at the transcription level of ISW1 or its target genes, qRT-PCR of ISW1 and transcriptome analysis of the wild-type and isw1Δ mutant strains treated with or without FLC was performed (Supplementary file 2). While the gene expression of ISW1 remains unchanged in the presence of drugs (Figure 1—figure supplement 1a), the expression of genes important in drug resistance (Denning and Bromley, 2015), including seven genes encoding ATP-binding cassette (ABC) transporters (CNAG_02296, CNAG_02430, CNAG_06338, CNAG_00792, CNAG_01575, CNAG_03600, and CNAG_02764), two genes encoding efflux proteins (CNAG_03101 and CNAG_01960), and three genes encoding major facilitator superfamily (MFS) transporters (CNAG_01674, CNAG_04898, and CNAG_06259), was significantly elevated in expression in the FLC-treated isw1Δ mutant (Figure 1c). Furthermore, we conducted a comparative analysis of the transcriptome data acquired from the wild-type and isw1Δ mutant strains in the absence of FLC treatment. The evidence presented indicates that the modulation of drug pumps by Isw1 is subject to the influence of FLC. This phenomenon can be attributed to the identification of modified gene expression in 11 specific genes within the isw1Δ mutant strain when exposed to FLC, while no alterations were observed in the absence of FLC. Nevertheless, a total of 10 genes displayed modified expression patterns, regardless of any administered drugs. The data collectively indicate that Isw1 plays a crucial role in regulating the drug efflux mechanism during drug treatment. In addition, in order to investigate the potential modulation of intracellular FLC concentration by Isw1, we employed high-performance liquid chromatography to quantify the levels of intracellular FLC. The results revealed a substantial reduction in FLC levels in the isw1Δ mutant compared to the wild-type strain (Figure 1d).

To further decipher 5-FC-resistance mechanisms of isw1Δ, we examined 5-FC-resistance pathways. As previously elucidated, C. neoformans employs two molecular processes for resistance to 5-FC and 5-FU (Billmyre et al., 2020; Loyse et al., 2013). In one, the purine-cytosine permease Fcy2 imports the prodrug 5-FC, which is then converted to toxic 5-FU by the cytosine deaminase Fcy1 (Figure 1e). On the other, the UDP-glucose dehydrogenase Ugd1 and the UDP-glucuronate decarboxylase Uxs1 participate in UDP-glucose metabolism, providing important functions in detoxifying 5-FU (Figure 1e). Therefore, gene expression alterations in FCY1, FCY2, FUR1, UGD1, and UXS1 were assayed using qRT-PCR (Figure 1f). The data showed that the expression of FCY1, FCY2, UGD1, and UXS1 was significantly reduced in isw1Δ when treated with 5-FC, suggesting a reduction in 5-FC uptake and conversion to toxic 5-FU (Figure 1f). These data suggested that Isw1 is a master transcriptional regulator of drug-resistance genes in C. neoformans.

Given the notable resemblance between cryptococcal Isw1 and the imitation switch chromatin remodeler found in S. cerevisiae (Chacin et al., 2023; Lin et al., 2019; Litwin et al., 2023), we sought to determine if the modulation of chromatin activity is the mechanism by which Isw1 regulates drug resistance. Initially, an ISW1-FLAG strain was generated by integrating the ISW1-FLAG construct into the original ISW1 allele of the isw1Δ strain, for the purpose of conducting chromatin immunoprecipitation tests (Figure 1—figure supplement 1b–d). The results revealed that the Isw1-Flag exhibited a significant binding affinity toward the promoter regulatory regions of many genes associated with drug resistance (Figure 1g). We then identified five distinct protein domains, including DNA-binding domains (DBD), SNF2, helicase C, HAND, and SLIDE domains in Isw1. Functions of these domains in DNA binding or chromatin remodeling processes were well established in S. cerevisiae (Grüne et al., 2003; Mellor and Morillon, 2004; Pinskaya et al., 2009; Rowbotham et al., 2011). We thereby generated truncation mutant alleles for these domains, specifically DBD, SNF2, or helicase C. The results showed that Isw1 function is significantly impaired (Figure 1h). Consistently, these truncation mutant strains exhibited significant resistance to the drug compounds, indicating the critical role of these domains in mediating Isw1 function (Figure 1h). In addition, we found that these truncation strains exhibited deficiencies in activating genes associated with drug resistance, mimicking the regulatory patterns observed in the isw1Δ strain (Figure 1i). Collectively, these data strongly supported the proposition that the chromatin remodeler Isw1 plays a pivotal role as a primary regulator of antifungal drug resistance in C. neoformans. This regulatory function is achieved through the direct interaction of Isw1 with its target genes and subsequently alteration of gene expression.

Characterization of cryptococcal Isw1-interacting proteins

The ISWI complex in S. cerevisiae typically comprises several protein components, such as Isw1, Ioc2, Ioc3, Ioc4, and Itc1. It functions in collaboration with Chd1, which has a substantial overlap in transcriptional target genes with Isw1 (Mellor and Morillon, 2004; Smolle et al., 2012; Sugiyama and Nikawa, 2001; Vary et al., 2003; Yadon et al., 2013). To further characterize cryptococcal ISWI functional mechanisms and regulatory pathways, we conducted a co-immunoprecipitation (co-IP) assay with Isw1-Flag, followed by mass spectrometry analysis (Supplementary file 4). We have found a total of 22 proteins that interacted with Isw1-Flag, and 11 are anticipated to be involved in the Isw1 network (Figure 2a), as shown by the STRING database (https://version-11-5.string-db.org). The results showed common Isw1-interacting proteins, including histones (such as histone H1/5, CNAG_02924) and Itc1. Because the Itc1 subunit has a notable function in the yeast ISWI complex (Sugiyama and Nikawa, 2001), we performed the protein–protein interaction between Isw1 and Itc1. As S. cerevisiae Isw1 acts in conjugation with Chd1 and Isw2’s DNA-binding ability has been implicated to be Sua7 dependent, these interactions were also tested (Smolle et al., 2012; Yadon et al., 2013). Similar to that of S. cerevisiae, Itc1 was also observed to engage in protein–protein interactions in C. neoformans (Figure 2b). Our mass spectrometry analysis failed to identify a homolog of Chd1 (Supplementary file 4). Nevertheless, the Chd1 homolog was found to co-immunoprecipitate with Isw1 in C. neoformans, whereas the Sua7 homolog did not exhibit the same manners (Figure 2c).

Characterization of Isw1-interacting proteins.

(a) Mass spectrometry analysis of Isw1 regulating network. Protein co-immunoprecipitation (co-IP) assays were carried out, followed by mass spectrometry analysis. Isw1-interacting proteins were identified, and interacting network was generated using StringDB. (b) Isw1 and Itc1 co-IP analysis. Proteins were isolated from strains expressing Isw1-FLAG or Itc1-HA or both, and co-IP followed by immunoblotting assays were performed. Antibodies against FLAG and HA epitope tags were used. (c) Isw1, Sua1, and Chd1 co-IP analysis. Proteins were isolated from strains expressing Isw1-FLAG, Isw1-FLAG/Chd1-HA, or Isw1-FLAG/Sua7-HA, and co-IP followed by immunoblotting assays were performed. Antibodies against FLAG and HA epitope tags were used. (d) Spotting analysis of chd1Δ and itc1Δ strains. Indicated strains were spotted onto YPD agar plates supplemented with antifungal agents. (e) Drug inhibitory tests of chd1Δ and itc1Δ strains. The H99, chd1Δ, and itc1Δ strains (n = 3 each) were tested to determine inhibitory effects of several antifungal agents. The experiments were performed as described in Figure 1b. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD).

We then investigated the extent to which the regulation of drug resistance by cryptococcal Isw1 is contingent upon the presence of these subunits. We created the mutants of itc1Δ and chd1Δ (Figure 2d, e) and found that itc1Δ cells were resistant to antifungal agents, but the chd1Δ strain demonstrated only limited drug resistance, which are not comparable to those observed in isw1Δ cells (Figure 2d, e). Therefore, these findings indicate that the control of Isw1 in drug resistance relies on the Isw1–Itc1 complex. However, given the presence of other Isw1-interacting proteins in our data, it is possible that Isw1 may potentially carry out its function through alternative complex regulatory networks.

Isw1 is an important in vivo drug-resistance regulator

We then sought to ask whether Isw1 plays a role in fungal pathogenicity and drug resistance in the context of cryptococcal infection and antifungal treatment. We gathered the evidence that total Isw1 protein levels are not affected by external stress inducers (Figure 3—figure supplement 1). In addition, we observed that the isw1Δ strain did not exhibit any growth impairment when subjected to stresses, such as changes in body temperature, osmotic stress, and stresses affecting the cell wall and membrane (Figure 3a). Moreover, a minor impairment in melanin production was observed, but no noticeable abnormalities in the development of the capsule were identified (Figure 3b, c). Next, cryptococcal intranasal infections were performed in BALB/c mice (Oliveira et al., 2021; Li et al., 2019). The results revealed no statistically significant changes in the survival rates and colony-forming units (CFUs) (14-day post infection) among the wild-type, isw1Δ, and ISW1 complemented strains (Figure 3d, e). Finally, mice were subjected to intranasal infection with either the wild-type or isw1Δ cells, followed by intraperitoneal administration of FLC or 5-FC. Pulmonary CFU quantification revealed that the isw1Δ strain exhibits enhanced fungal burdens when treated with both drugs (Figure 3e). This enhancement is statistically significant, as indicated by the p-values of less than 0.0001 (p < 0.0001). These data thus provided substantial evidence supporting the pivotal role of Isw1 as a key regulator of drug resistance, both in vivo and in vitro.

Figure 3 with 1 supplement see all
Isw1 plays a critical role in drug resistance during pulmonary infection.

(a) Spotting analysis of isw1Δ in stress conditions. H99, isw1Δ, and ISW1 complementation strains were spotted onto YPD agar plates supplemented with indicated chemicals. (b) Melanin formation of the isw1Δ strain. (c) Capsule formation and quantification of the isw1Δ strain. H99 and isw1Δ cells were induced for capsule structure. Capsular sizes were quantified. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (d) Animal survival analysis and the Kaplan–Meier survival curves of wild-type and isw1Δ. Significance was determined using a log-rank (Mantel–Cox) test. (e) Colony-forming unit (CFU) analysis of the isw1Δ strain. Mice (n = 10 each) were infected with H99 or isw1Δ cells. At 7-day post infection, animals were treated with phosphate-buffered saline (PBS), or 5 or 45 mg/kg of fluconazole (FLC), or 100 or 200 mg/kg 5-fluorocytosine (5-FC). Animals were treated with drugs in a 24-hr interval on a daily basis. At 14-day post infection, lung tissues were removed and homogenized. CFUs were performed. Data are expressed as mean ± standard deviation (SD). Two-tailed unpaired t-tests were used for H99-PBS and isw1Δ-PBS group. Two-way analyses of variance (ANOVAs) were used for drug-treated groups.

Isw1 undergoes protein degradation in the presence of azole compounds

Because Isw1 governs the expression of multiple genes required for azole resistance and the ISW1 gene expression was not reduced in the presence of antifungal drugs (Figure 1—figure supplement 1a), we examined changes in protein stability as a response to antifungal agents. Using cycloheximide to inhibit protein synthesis, the Isw1-Flag fusion protein stability was found to decrease gradually in concentration- and time-dependent manner upon exposure to FLC (Figure 4a, b). Specifically, a reduction of 50% was observed 30 min after FLC exposure. Similarly, 5-FC exposure also reduced Isw1-Flag levels (Figure 4c, d). Collectively, these results demonstrated that C. neoformans actively reduces Isw1 protein levels through protein degradation in the presence of antifungal drugs.

Isw1 is an acetylated and ubiquitinated protein.

(a) Immunoblotting analysis. The ISW1-FLAG strain was preincubated with 200 μM cycloheximide (CHX) for 1 hr followed by exposure to various concentrations of fluconazole (FLC) for 0.5 hr. Anti-Flag and anti-histone 3 antibodies were used. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (b) Immunoblotting analysis. The ISW1-FLAG strain was preincubated with 200 μM cycloheximide (CHX) for 1 hr followed by exposure to 40 μg/ml FLC for 5, 15, or 30 min. Cells not exposed to FLC but held for 30 min were used as a negative control. Anti-Flag and anti-histone 3 antibodies were used. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD. (c) Immunoblotting analysis. Testing and data treatment were exactly as described for Figure 2a with the exception that 5-fluorocytosine (5-FC) was used as the antifungal agent. (d) Immunoblotting analysis. Testing and data treatment were exactly as described in Figure 2b, except that 5-FC was used as the antifungal agent. (e) Ubiquitin analysis of Isw1 via mass spectrometry. The Isw1-Flag proteins were pulled down and analyzed for ubiquitination. Results for Isw1K441Ub are shown. (f) Schematic of Isw1 showing acetylation (Li et al., 2019) and ubiquitination sites.

We then hypothesized that Isw1 degradation might be via a ubiquitin–proteasome pathway in response to antifungal drugs. To test this, the Isw1-Flag protein was immunoprecipitated and then analyzed using mass spectrometry to identify putative ubiquitination PTM sites. Consistent with our hypothesis, Isw1 is ubiquitinated (Figure 4e) at 15 sites (Figure 4f). These results indicated that the ubiquitination machinery of Isw1 is actively initiated during drug exposure, and this, in turn, decreases Isw1 protein levels and hinders Isw1 transcription repression of genes for drug resistance. The finding of Isw1 subject to ubiquitination and acetylation regulation also suggested that there exists an interplay network simultaneously controlling Isw1 stability in response to antifungal drugs. We then set forth to address whether there is a PTM interplay between acetylation and ubiquitination in Isw1.

Acetylation of Isw1K97 (Isw1K97ac) is essential for protein stability

To dissect the interplay between acetylation and ubiquitination in Isw1, we examined the role of acetylation in modulating Isw1 function by determining acetylation levels responding to antifungal drugs. The presence of antifungal agents strongly repressed acetylation levels, in contrast to deacetylation inhibitors trichostatin A (TSA) and nicotinamide (NAM), which enhanced acetylation levels (Figure 5a, b). These data suggested a positively regulated deacetylation process in Isw1 in response to antifungal drugs. To decipher this regulation mechanism more, three acetylation sites, K89, K97, and K113, located to the DBD were mutated to arginine (R) to mimic a deacetylated status or to glutamine (Q) to mimic acetylated Isw1 (Figure 4f). Gene copy numbers and transcription levels were confirmed to be equivalent to those of the wild-type strain (Figure 5—figure supplement 1a, b). Triple-, double-, and single-mutated strains were generated, and their drug-resistance phenotypes were compared. Of the triple-mutated strains, cells with three R mutations demonstrated drug-resistant growth phenotypes that were similar to those of the isw1Δ strain, whereas those with three Q mutations showed the wild-type growth in the presence of antifungal drugs (Figure 5c). Double-mutated strains, ISW1K89R, K97R and ISW1K97R, K113R, showed resistance to antifungal drugs, wherein the ISW1K97R, K113R strain showed less resistance than ISW1K89R, K97R and the isw1Δ strain (Figure 5—figure supplement 1c). These data suggested that the acetylation status of Isw1K97 is important in conferring drug resistance. Of the strains that have single-R mutation, the ISW1K97R strain showed robust resistance to antifungal drugs, mimicking the isw1Δ strain (Figure 5d). Interestingly, the ISW1K97Q strain showed no drug resistance (Figure 5—figure supplement 1d). Collectively, these data strongly demonstrated that the acetylation status of Isw1K97 plays a critical role in regulating Isw1 protein stability and function in response to antifungal drugs.

Figure 5 with 1 supplement see all
The acetylation status of Isw1K97 (Isw1K97ac) is essential in Isw1 protein stability.

(a) Acetylation analysis of Isw1. Cells were treated with 3 μM trichostatin A (TSA), 20 mM nicotinamide (NAM), and fluconazole (FLC). The Isw1-Flag proteins were pulled down, and immunoblotting assays were performed using anti-Kac and anti-Flag antibodies. (b) Acetylation analysis of Isw1. Cells were treated with TSA, NAM, and 5-fluorocytosine (5-FC). The Isw1-Flag proteins were pulled down, and immunoblotting assays were performed using anti-Kac and anti-Flag antibodies. (c) Spotting assays of ISW1 mutants. The ISW1K89R, K97R, K113R and ISW1K89Q, K97Q, K113Q strains were tested for drug resistance. (d) Spotting assays of ISW1 mutants. The ISW1K89R, ISW1K97R, and ISW1K113R strains were tested for drug resistance. (e) Immunoblotting assays of ISW1 mutants. The wild-type, ISW1K89R, K97R, K113R, and ISW1K89Q, K97Q, K113Q strains were tested for Isw1 levels. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (f) Immunoblotting assays of ISW1 mutants. The wild-type, ISW1K89R, ISW1K97R, and ISW1K113R strains were tested for Isw1 levels. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD. (g) Immunoblotting assay of Isw1. The wild-type strain was preincubated with 200 μM cycloheximide for 1 hr. Proteins were isolated at indicated time points. Three biological replicates of immunoblotting were performed, and results were used for quantification. One-way analysis of variance (ANOVA) was used. (h) Immunoblotting assay of Isw1K97Q. The analysis was performed as described in Figure 3g. (i) Immunoblotting assay of Isw1K97R. The analysis was performed as described in Figure 3g. (j) Comparisons of assay results to determine Isw1 stability. The relative intensities from the results shown in Figure 3g–i were plotted. Two-way ANOVA was used. Data are expressed as mean ± SD. (k) Analyses of drug-resistance genes using qRT-PCR. Samples of RNA (n = 3) were isolated from ISW1K97Q and ISW1K97R treated with FLC or 5-FC. Representative drug-resistance genes were quantified using qRT-PCR. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD.

To further investigate how Isw1 degradation correlates with drug resistance of C. neoformans, we tested how Isw1K97 acetylation affects its degradation using the immunoblotting method, and the results showed that triple-R mutation resulted in a significant reduction in levels of Isw1-Flag. Meanwhile, triple-Q mutation resulted in Isw1-Flag levels comparable to wild-type strains (Figure 5e). Similarly, the single site mutant, ISW1K97R, showed a lower level of Isw1-Flag (Figure 5f). In contrast, no changes were observed for Isw1K97Q (Figure 5—figure supplement 1e). Moreover, protein levels of wild-type Isw1 and mutated Isw1K97R gradually diminished over time. While those of Isw1K97Q remained constant (Figure 5g–i), a faster degradation was observed for Isw1K97R (Figure 5j). Therefore, Isw1K97 is an essential regulation site responsible for Isw1 stability; that is, acetylation at K97 blocked the degradation of Isw1, and deacetylation at K97 facilitated Isw1 degradation. Finally, analysis of Isw1 target gene expression in Isw1K97 mutation strains demonstrated significantly the increased expression of transporter genes and the decreased expression of 5-FC-resistance genes (Figure 5k). These findings were consistent with the results of transcriptome and qRT-PCR analyses in the isw1Δ strain (Figure 1c, f).

The interplay between acetylation and ubiquitination governs Isw1 degradation

As proteins undergo degradation via autophagy and proteasomal pathways, we employed autophagy inhibitor rapamycin and proteasome inhibitor MG132 to investigate Isw1 degradation. Immunoblotting results showed that rapamycin and MG132 promote Isw1-Flag levels, with rapamycin’s effect less prominent than MG132 (Figure 6a). These findings suggested that both degradation pathways are utilized in Isw1-Flag degradation, but the ubiquitin-mediated proteasomal process has a more predominant role. Additionally, we tested whether antifungal agents could induce protein degradation when the proteasome pathway is blocked. Cells treated with MG132 and FLC or 5-FC yielded slightly different results. Isw1-Flag protein levels were unaffected in cells treated with FLC, but the levels were reduced with 5-FC (Figure 6b, c).

Figure 6 with 1 supplement see all
Isw1K97ac is critical for Isw1 ubiquitin–proteasome degradation.

(a) Immunoblotting assays of Isw1-Flag. Proteins Isw1K97Q, Isw1K97R, and Isw1 were tested, where the wild-type stain was incubated with 200 μM MG132 and 5 nM rapamycin for 10 hr. Three biological replicates of the assays were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (b) Immunoblotting assays of Isw1-Flag. Testing and data treatment were exactly as described in Figure 4a, except that the wild-type sample was treated with 200 μM of MG132 and 40 μg/ml fluconazole (FLC). (c) Immunoblotting assays of Isw1-Flag. Testing and data treatment were exactly as described in Figure 4a, except that the wild-type sample was treated with 200 μM MG132 and 400 μg/ml 5-fluorocytosine (5-FC). (d) Immunoblotting assays of Isw1K97Q and Isw1K97R. Proteins were either treated with MG132 or not before testing. Three biological replicates of the assays were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD. (e) Spotting assays of ISW1 acetylation and ubiquitination mutants. Indicated strains were spotted onto YPD agar either supplemented with an antifungal agent or left blank. (f) Immunoblotting assays of ISW1 acetylation and ubiquitination mutants. Protein samples were isolated from the indicated ISW1 mutants. Immunoblotting assays were performed. (g) Quantification of immunoblotting results. The immunoblotting assays described for Figure 3f were performed using three independent samples, and the results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD.

Given that K97 deacetylation could trigger hyper-ubiquitination of Isw1, we analyzed Isw1 ubiquitination sites and their regulation mechanisms by K97 acetylation levels and found that MG132 treatment results in a more robust enhanced in Isw1K97R-Flag levels (Figure 6d). We further performed ubiquitination site mutations in the genetic background of ISW1K97R, and sites neighboring K97 were selected for mutagenesis. We found that, of the seven ubiquitination sites (Figure 6—figure supplement 1a, b), five (K98, K147, K183, K347, and K415) failed to affect drug-resistant growth phenotypes of the ISW1K97R mutant (Figure 6—figure supplement 1c). Only ISW1K113R and ISW1K441R mutations exhibited reduced drug-resistant growth of the ISW1K97R stain (Figure 6e), indicating that they affect drug resistance by modulating Isw1 protein stability in the absence of acetylation at the K97. The immunoblotting analysis further showed that, while all ubiquitination mutants exhibited moderately enhanced Isw1-Flag levels (Figure 6f, g), robust elevations were detected in Isw1K97R, K113R (5.6-fold) and Isw1K97R, K441R (14.5-fold) (Figure 6f, g). Interestingly, the K113 site may undergo acetylation or ubiquitination modifications, whereas the K441 site undergoes only ubiquitination. Collectively, these results showed that Isw1K113 and Isw1K441 provide a predominant role in regulating the ubiquitin-proteasome process of Isw1 and that acetylation at Isw1K97 has a broad role in controlling the ubiquitination process at those sites.

The acetylation status of Isw1K97 modulates the binding of an E3 ligase to Isw1

To analyze the molecular mechanism by which Isw1K97 regulates the ubiquitin–proteasome process, we generated nine knockout mutants of E3 ligase encoding genes in the genetic background of ISW1K97R and identified that Cdc4 is an E3 ligase for Isw1 (Figure 7—figure supplement 1a and Figure 7a). We found that the ISW1K97R/cdc4∆ strain becomes sensitive to antifungal agents (Figure 7a). In addition, disrupting CDC4 in the H99 strain does not affect the growth of cells in response to antifungal drugs (Figure 7—figure supplement 1b). Immunoblotting showed a strong elevation in Isw1K97R-Flag levels in the ISW1K97R/cdc4∆ strain (Figure 7b), in contrast to the ISW1K97R/fwd1∆ strain (Figure 7c), suggesting a potential interaction between Cdc4 and Isw1. We then performed a co-IP assay and found that Cdc4-HA co-precipitates with Isw1-Flag (Figure 7d).

Figure 7 with 1 supplement see all
IswK97ac blocks the binding of Isw1 to the E3 ligase Cdc4.

(a) Spotting assays of E3 ligase mutants. Indicated strains were spotted onto YPD agar either supplemented with an antifungal agent or left blank. (b) Immunoblotting assays of cdc4Δ. Protein samples were isolated from isw1Δ/ISW1K97R/cdc4Δ and its relevant control strains. Three independent samples were tested and quantified. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (c) Immunoblotting assays of fwd1Δ. Protein samples were isolated from isw1Δ/ISW1K97R/fwd1Δ and its relevant control strains. Three independent samples were tested and quantified. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD. (d) Protein co-immunoprecipitation (co-IP) of Cdc4 and Isw1. Protein samples were isolated from the strain expressing Cdc4-HA and Isw1-Flag, and co-IP was performed. (e) Protein co-IP of Cdc4 and Isw1 K97 mutant proteins. Protein samples were isolated from the strain co-expressing Cdc4-HA and Isw1K97R-Flag and the strain co-expressing Cdc4-HA and Isw1K97Q-Flag. Co-IP was performed for each.

We also carried out co-IP to examine interactions between Cdc4-HA and Isw1K97R and Cdc4-HA and Isw1K97Q, and the results showed an interaction between Cdc4 and Isw1K97R but a reduction in the strength of the interaction between Cdc4 and Isw1K97Q, indicating the acetylation of Isw1K97 hinders its binding of the Cdc4 E3 ligase (Figure 7e). These data provided convincing evidence that K97 acetylation is a key player in modulating ubiquitin–proteasome degradation of Isw1.

The Isw1–proteasome regulation axis promotes drug resistance in clinical isolates

We have presented empirical evidence that highlights the significant involvement of Isw1 and the acetylation–Isw1–ubiquitination regulatory pathway in the modulation of drug resistance in C. neoformans. In order to assess the potential wide-ranging applicability of this regulatory function, a random selection of 18 clinical isolates of C. neoformans was subjected to testing (Supplementary file 1). Among them, a total of 12 isolates (CDLC4, CDLC13, CDLC15, CDLC25, CDLC60, CDLC61, CDLC62, CDLC98, CDLC125, CDLC135, CDLC141, and CDLC150) exhibited significant resistance to at least one antifungal drug. It is noteworthy that the application of PCR in conjunction with sequencing analysis has yielded results indicating the absence of mutations in the drug-resistance genes of these clinical isolates (Supplementary file 5). Conversely, six isolates (CDLC120, CDLC6, CDLC27, CDLC37, CDLC43, and CDLC100) demonstrated no resistance to antifungal agents, and their growth patterns resembled that of the H99 strain in the presence of these drugs (Figure 8—figure supplement 1a). Subsequently, a 3′ integrative FLAG-tag construct was introduced into multiple clinical strains, enabling the transcription of Isw1-Flag under the control of the ISW1 endogenous promoter (Sun et al., 2014). The protein expression results exhibited three distinct classes: (1) Strains that displayed phenotypes of multidrug resistance exhibited notably diminished levels of Isw1-Flag. These strains include CDLC15, CDLC25, CDLC61, CDLC62, and CDLC98. (2) Conversely, strains that exhibited sensitivity to drug treatment displayed robust protein levels of Isw1-Flag. Notable examples of such strains are CDCL120, CDCLC6, CDCL37, CDCLC43, and CDLC100. (3) Drug-resistant strains demonstrated Isw1-Flag levels comparable to those of the H99 strain. This similarity was observed in strains such as CDCL141 and CDLC4 (Figure 8a and Figure 8—figure supplement 1b). Significantly, the changes in Isw1-Flag levels observed were not attributed to transcriptional modifications (Figure 8—figure supplement 1c).

Figure 8 with 1 supplement see all
Clinical C. neoformans isolates show Isw1-mediated drug-resistance phenotypes.

(a) Immunoblotting assays of Isw1-Flag from clinical isolates. Protein samples were isolated from H99 and clinical isolates expressing Isw1-Flag. Immunoblotting analyses were performed. (b) Immunoblotting analyses of Isw1 levels under MG132 treatment. Cells were treated with MG132, then immunoblotting assays were performed on Isw1-Flag using anti-Flag antibodies. (c) Three independent repetitions from Figure 8b were performed, and results were used for quantification. Two-tailed unpaired t-tests were used. Data are expressed as mean ± standard deviation (SD). (d) Immunoblotting analyses of Isw1 acetylation in clinical strains. Cells were treated with trichostatin A and nicotinamide, then Isw1-Flag was immunoprecipitated. Acetylation levels were examined using anti-Kac antibodies. (e–i) Drug inhibitory analyses of Isw1-overexpressing clinical strains. Clinical strains harboring integrative overexpressing plasmid of ISW1K97Q were tested for drug resistance, and minimum inhibitory concentrations (MICs) were determined. OE indicates strains with overexpressed ISW1K97Q. The experiments were performed as described in Figure 1b. Quantitative data were depicted in color (see color bar) and bar plots. Two-tailed unpaired t-tests were used. Data are expressed as mean ± SD.

We then determined whether the Isw1 of clinical isolates exhibit a comparable PTM regulation pattern as observed in the H99 strain. In a manner akin to PTMs observed in Isw1 from the H99 strain, our analysis of samples derived from three distinct clinical isolates revealed heightened levels of Isw1 upon exposure to MG132 (Figure 8b, c). Furthermore, the clinical isolates that were subjected to TSA and NAM treatment exhibited enhanced levels of Isw1 acetylation, as demonstrated in (Figure 8d). The clinical isolates were subsequently analyzed to ascertain if drug resistance arises due to alterations in Isw1 protein levels subsequent to transformation with an integrative plasmid that overexpresses the non-degradable variant of Isw1, known as Isw1K97Q. Initially, the H99 strain was subjected to testing, which revealed a decrease in cell growth when the ISW1K97Q gene was overexpressed in the presence of antifungal drugs (Figure 8e). Subsequently, four strains, namely CDLC120, CDLC141, CDLC135, and CDLC61, were subjected to transformation and subsequent testing. The results indicated that all four clinical strains overexpressing ISW1K97Q displayed a susceptible cell growth response to antifungal drugs (Figure 8f–i). Conversely, the CDLC135 ISW1K97Q strain exhibited a sensitive growth pattern toward azoles but demonstrated a resistant growth phenotype when exposed to 5-FC (Figure 8g). While the Isw1 level remains constant in CDLC141, an elevation in Isw1 level was found to suppress the growth of drug resistance in this strain (Figure 8h). Hence, the acetylation–Isw1–ubiquitination regulatory axis represents a naturally occurring mechanism employed to modulate multidrug resistance in clinical strains of C. neoformans.

Discussion

Fungi have developed sophisticated machinery to combat various stress inducers, and the rapid emergence of resistance to antifungal agents is one of the major factors in the failure of clinical therapies for fungal infections (Denning and Bromley, 2015). A typical tactic used by fungi to overcome antifungal toxicity is to utilize polymorphisms or mutations in drug targets or their regulatory components. Clinical polymorphisms were widely shown in drug targets, such as ergosterol biosynthesis and its transcription regulatory process (Denning and Bromley, 2015; Hu et al., 2017). However, unlike immediate intracellular responses, the accumulation of mutations or polymorphisms in drug resistance is a somewhat delayed process that frequently develops over a series of cell divisions. Acetylation and ubiquitination are critical modulators of protein activities or stabilities in fungi that enable rapid intracellular adaptations to environmental or chemical stressors (Li et al., 2019; Wu et al., 2021), but the underlying mechanisms are unclear. Recently, a study showed that the deactivation of a heat-shock Hsp90 client protein and its stability due to changes in protein acetylation impacts drug resistance in C. albicans (Robbins et al., 2012). Additional studies have demonstrated that the catalytic subunit of the histone acetyltransferase, notably Gcn5, controls biofilm formation, morphology, and susceptibility to antifungal drugs in several fungi (O’Meara et al., 2010; Rashid et al., 2022; Yu et al., 2022). Despite this, knowledge of the molecular machinery of PTMs in modulating drug resistance remains not clear.

We demonstrated that the chromatin remodeler Isw1 is a master regulator of drug resistance in C. neoformans, and the acetylation–Isw1–ubiquitination axis is crucial in modulating the expression of multiple drug-resistance genes. In S. cerevisiae, Isw1 is a key component of the ISWI complex capable of forming complexes with Ioc2, Ioc3, Ioc4, and Itc1 to modulate transcription initiation and elongation (Mellor and Morillon, 2004; Smolle et al., 2012; Sugiyama and Nikawa, 2001; Vary et al., 2003; Yadon et al., 2013), and S. cerevisiae Isw1 and Chd1 exhibit functional overlap in transcription within the Set2 pathway (Smolle et al., 2012). Additionally, we ascertain the involvement of subunits of the ISWI complex-related protein in the regulation of drug resistance. Although no IOC genes of the yeast Isw1 regulatory machinery are found in the C. neoformans genome (https://fungidb.org/fungidb/app), we demonstrated that Chd1 and Itc1 have the ability to form a protein complex with Isw1. Interestingly, the disruption of ITC1, but not CHD1, resulted in multidrug resistance in C. neoformans. This finding provides additional evidence that the regulatory mechanisms of the cryptococcal ISW1 complex in drug resistance are mediated by the Isw1–Itc1 regulatory axis, rather than the Isw1–Chd1 pathway.

In C. neoformans, transcriptome analysis revealed 1275 genes, approximately 18.3% of the genome, that were significantly differentially expressed in the isw1Δ mutant when treated with FLC. The changes in gene expression had a significant impact on the drug efflux system through the activation of 12 drug pump genes and the repression of 9 pump genes, which included ABC and MFS transporter genes. The results presented suggests that the strong drug-resistance phenotype observed in the isw1Δ strain may be attributed to the concurrent modulation of gene expression of multiple drug pumps, which subsequently facilitate the active removal of intracellular drug molecules. The expressions of genes required for resisting 5-FC and 5-FU were reduced when cells were treated with 5-FC. While Isw1 represses genes responsible for resistance to FLC, it also positively modulates the expression of genes required for 5-FC resistance, implying that chromatin remodeling of Isw1 is necessary as the cell responds to disparate chemical stresses and that Isw1 engages distinct remodeling venues to overcome drug toxicity. The overall abundance of Isw1 remains constant in response to external stimuli, and alterations in Isw1 protein levels may change when protein synthesis is inhibited, due to the interplay between two PTMs. Nevertheless, the investigations on the growth of cells demonstrated that the isw1Δ strain exhibited the ability to endure stresses, so indicating that Isw1 does not regulate fungal virulence factors and pathogenicity. The fungal burden analysis provided further evidence by demonstrating comparable amounts of fungal colonization in both the wild-type and isw1Δ strains. However, the isw1Δ strain displayed notable resistance to antifungal treatments both in vitro condition and in animal models. The findings provide strong evidence for the pivotal function of Isw1 in the development of drug resistance in C. neoformans during infection, and revealed a previously unidentified controller of drug resistance in fungi.

We also showed that the Isw1 protein and its acetylation level act reciprocally to govern drug resistance in C. neoformans (Figure 9). This was confirmed by uncovering the interplay mechanism between acetylation and ubiquitination. The total acetylation levels of Isw1 were reduced when cells were treated with FLC or 5-FC, leading to the activation of Isw1 ubiquitination machinery. We identified that the K97 acetylation site functions as the essential regulating component of this interplay. We also found that K97 acetylation acts as a switch for ubiquitin conjugation proceeding proteasome-mediated degradation. When deacetylated, K97 triggers the activation of Isw1 degradation via the ubiquitin–proteasome process (acetylated K97 blocks the physical interaction with Cdc4). Compared to the ISW1K97R strain, ISW1K97R/cdc4Δ was sensitive to antifungal agents; however, it showed moderately resistant growth in comparison to the wild-type strain. These data suggested that Isw1 could also be modulated by other proteins, such as another uncharacterized E3 ligase. Such hypothesis is supported by evidence from the comparison of Isw1 ubiquitination mutants with those of the ISW1K97R/cdc4Δ strain, and from that, the Isw1K441R mutant had a 14.5-fold increase of Isw1 and a 2-fold increase in the ISW1K97R/cdc4Δ strain.

A model of the mechanism of Isw1 posttranslational modification (PTM) interaction in C. neoformans drug resistance.

In a drug-free environment, acetylated Isw1 regulates 5-fluorocytosine (5-FC)-resistance gene expression and represses drug pump gene expression. Azoles and 5-FC trigger the deacetylation process at the K97 residue of Isw1, initiating the ubiquitin-mediated proteasomal degradation of Isw1 through the E3 ligase Cdc4. The decrease in Isw1 protein level results in the stimulation of drug pump gene expression and the inhibition of 5-FC-resistance genes.

The identified ubiquitination sites were classified into three groups based on the domination of Isw1 stability regulation: predominant, moderate, and minor. While Isw1K441 was a predominant regulating site of ubiquitination that extensively modulates Isw1 protein degradation and drug resistance, Isw1K147, Isw1K183, Isw1K347, and Isw1K415 played a minor role in Isw1 degradation and no roles in drug resistance. Interestingly, Isw1K113 played a moderate role in ubiquitination-mediated Isw1 degradation and was identified to have both an acetylation site and a ubiquitination site. A single mutation at Isw1K113 had no effect on Isw1 protein levels since K97 acetylation that prevented Cdc4 binding remains intact. In the strain background of degradable Isw1 mutant (Isw1K97R), the K113R mutation was capable of protecting from degradation, and its protein levels had an increase of 5.6-fold, allowing the ISW1K97R, K113R strain to be drug resistant. These data suggested that the K113 site contributes to Isw1 protein stability when the degradation of Isw1 process is initiated. Although it is challenging to dissect the function of acetylation and ubiquitination at Isw1K113, drug treatment represents a deacetylation process for Isw1, and deacetylated Isw1K113 is most likely ubiquitinated.

The examination of clinical isolates has uncovered a widespread regulatory phenomenon involving Isw1 in the context of drug resistance. In general, clinical isolates exhibiting higher levels of antifungal resistance were shown to have correspondingly reduced expression of Isw1. While certain resistant isolates, including CDCL141, exhibited unaltered endogenous levels of Isw1, the strain’s drug-resistance phenotype is contingent upon the levels of Isw1, as evidenced by the heightened sensitivity to antifungal agents upon overexpression of Isw1. Furthermore, acetylation and ubiquitination were detected in the clinical isolates examined, indicating that Isw1 undergoes acetylation and is targeted by the ubiquitin–proteasome system. Significantly, the overexpression of Isw1 was found to mitigate resistance to drugs in clinical isolates. The strain CDLC135 exhibited a reciprocal regulation pattern in response to 5-FC, suggesting that the process of 5-FC resistance is indeed controlled by Isw1, but in a disparate manner from other isolates. It is probable that the Isw1 regulation axis has undergone rewiring in these clinical isolates. However, it was shown that the CDLC135 strain overexpressing Isw1 exhibited sensitivity toward two azole drugs. The findings as mentioned above indicated that the modulation of Isw1 protein levels has a role in drug resistance observed in clinical isolates.

Taken together, our evidence demonstrates the critical function of Isw1 as a master regulator of multidrug responses in both laboratory and clinical strains. It allows us to decipher the molecular mechanism of the acetylation–Isw1–ubiquitination axis that modulates the expression of drug-resistant genes. These findings underscore the importance of performing thorough evaluations of PTMs in drug-resistance mechanism studies, highlighting a potential strategy for overcoming fungal drug resistance.

Materials and methods

Strains and growth conditions

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Fungal cells (Supplementary file 1) were routinely grown in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose). The biolistic transformation was performed using a YPD medium supplemented with 100 μg/ml nourseothricin (WERNER BioAgents), 200 μg/ml neomycin (Inalco), and 200 U/ml hygromycin B (Calbiochem) followed by colony selection. Deacetylases were blocked using 3 μM TSA (MedChemExpress) and 20 mM NAM (Sigma). Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress). The melanin synthesis was conducted by culturing it on L-DOPA agar medium at a temperature of 37°C. C. neoformans capsules were conducted in Dulbecco’s modified Eagle medium, which was supplemented with 10% fetal bovine serum.

Fungal proliferation in response to antifungal agents

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The measurement of antifungal susceptibility was conducted using a dose–response method as previously described (Iyer et al., 2023; Revie et al., 2022), utilizing 96-well microtitre plates. In brief, dose–response tests were conducted using twofold serial dilutions of FLC (MedChemExpress), KTC (MedChemExpress), or 5-FC (MedChemExpress) in a final volume of 100 µl. Overnight YPD cultures were washed three times with phosphate-buffered saline (PBS) and diluted to an optical density of 0.02 at 600 nm. Following this, 100 µl of the resulting cell suspension was carefully dispensed into individual wells of a 96-well plate, with each well containing approximately 10,000 cells. The well plate was subjected to incubation at a temperature of 30°C for either 24 or 48 hr. Subsequently, optical density measurements at a wavelength of 600 nm were obtained using a Synergy HTX microplate reader manufactured by BioTek, as previously described (Billmyre et al., 2020). The growth of the relevant strain was standardized by normalizing it to the well without drug treatment. The average optical density values of technical duplicate measurements are depicted in the plot. Three biological replicates were conducted for each strain. The quantitative representation of dose–response assay results was visually shown using the software Java TreeView 1.1.3 (http://jtreeview.sourceforge.net) with a contrast value of 0.5. To facilitate the growth of dose–response assays, growth ratios were established for a minimum of three biological replicates. The data were graphed utilizing GraphPad Prism software.

Chromatin immunoprecipitation and PCR

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Co-IP of Isw1-Flag was performed as previously described (Gao et al., 2022). ISW1-FLAG was cultivated in YPD media overnight and subcultured to an A600 of 0.8 (mid-log phase). 50 ml of the cell culture was added to 200 ml conical flasks containing 1% formaldehyde, and the flasks were incubated at room temperature with moderate rocking for 15 min. To end the cross-linking reaction, 2.5 M glycine was added to the mixture, which was then held for 5 min. At 4°C, cells were extracted at 1000 × g and washed twice with ice-cold PBS containing 125 mM glycine. After the chromatin is extracted, the DNA fragments are broken by ultrasound. The ultrasound condition is 3% power for 5 min (run for 3.5 s and then pause for 3 s). The fragment of the genome was then purified. IgG was used as the negative control for chromatin immunoprecipitation-qPCR. Quantitative real-time PCR (CFX96 real-time instrument; Bio-Rad) was used to analyze the gene abundance of immunoprecipitation using the specific primer pairs shown in Supplementary primers.

Mass spectrometry

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Mass spectrometry was performed to analyze Isw1 ubiquitination. The ISW1-FLAG complementation strains were subcultured at 30°C with 200 μM MG132 (MedChemExpress) in 50 ml YPD media at 30°C, and cells in the mid-log phase were used. Cell proteins were extracted using lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% NP-40; pH 7.5) with 1× protease inhibitor cocktail (CWBIO) and 40 mM phenylmethylsulfonyl fluoride(PMSF). All lysed protein samples were incubated with anti-Flag magnetic beads (Sigma) at 4°C overnight. The beads were washed with Tris-Buffered Saline (TBS) buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100; pH 7.4) three times, and the bound proteins were extracted into protein loading buffer (125 mM Tris–HCl, 4% sodium dodecyl sulfate [SDS], 20% glycerol; pH 7.5) at 95°C for 5 min. All protein samples were separated using 8% SDS–polyacrylamide gel electrophoresis (PAGE), and the protein gel was stained with Coomassie Brilliant Blue R-250 (BBI Life Sciences) followed by clipping the gel strip.

In-gel tryptic digestion was performed by destaining the gel strips in 50 mM NH4HCO3 in 50% acetonitrile (vol/vol) until clear. The gel strips were dehydrated using 100 μl 100% acetonitrile for 5 min, rid of liquid, rehydrated in 10 mM dithiothreitol, then incubated at 56°C for 60 min. They were again dehydrated in 100% acetonitrile and rid of liquid, then rehydrated with 55 mM iodoacetamide followed by incubation at room temperature in the dark for 45 min. Next, they were washed with 50 mM NH4HCO3, dehydrated with 100% acetonitrile, then rehydrated with 10 ng/μl trypsin and resuspended in 50 mM NH4HCO3 on ice for 1 hr. After removing excess liquid, they were digested in trypsin at 37°C overnight, then peptides were extracted using 50% acetonitrile/5% formic acid followed by 100% acetonitrile.

The peptides were dried completely and then resuspended in 2% acetonitrile/0.1% formic acid. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) then loaded directly onto a homemade reversed-phase analytical column (15 cm × 75 μm) on an EASY-nLC 1000 UPLC system. They were eluted at 400 nl/min using a gradient mobile phase that increased in solvent B (0.1% formic acid in 98% acetonitrile) from 6% to 23% over 16 min, from 23% to 35% over 8 min and from 35% to 80% over 3 min. Elution continued at 80% for an additional 3 min.

The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) using a Q Exactive Plus (Thermo) mass spectrometer coupled to the UPLC. The electrospray voltage applied was 2.0 kV, the m/z scan range was 350–1800 for a full scan, and intact peptides were detected using an Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using an NCE of 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. The data-dependent procedure alternated between 1 MS scan and 20 MS/MS scans with a 15.0-s dynamic exclusion. The automatic gain control was set at 5E4.

The resulting MS/MS data were processed using Proteome Discoverer 1.3. Spectra were compared against acetylation, ubiquitination, or sumoylation databases. Trypsin/P (or other enzymes, if any) was specified as a cleavage enzyme, allowing up to four missing cleavages. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Fixed modification was set to cysteine alkylation; variable modifications were set to lysine acetylation, ubiquitination, or sumoylation (QEQQTGG and QQQTGG), methionine oxidation, and protein N-terminal acetylation. Peptide confidence was set to ‘high’, and peptide ion score was set to ‘>20’.

Strain generation

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C. neoformans mutants were generated using the H99 strain and biolistic transformation (Toffaletti et al., 1993). The neomycin- or nourseothricin-resistance marker was amplified using primers M13F and M13R (Supplementary file 3). The upstream and downstream DNA sequences of the target gene and the selective marker sequence were joined using overlapping PCR. The resulting PCR fragments were purified, concentrated, and transformed into the H99 strain using biolistic transformation. Transformants were selected on YPD agar supplemented with either neomycin or nourseothricin. Correct integration and the loss of target DNA sequences were confirmed using diagnostic PCR.

The ISW1 gene (CNAG_05552) was disrupted by the homologous replacement of its open reading frame (ORF) with a piece of DNA containing a dominant drug-resistance gene marker as described henceforth. In the first round of PCR, the primer pairs 3534/3535 and 3536/3537 were used to amplify the 5′ and 3′ flanking regions, respectively, of the ISW1 gene. The gel-extracted DNA fragments from the first round of PCR were used as templates, and the isw1∆::NEO construct was amplified using the 3534/3537 primer pair. The H99 strain of C. neoformans was biolistically transformed with the deletion allele. To identify the desired isw1∆ mutant, diagnostic PCR was performed using the 3534/3537 primer pair, and real-time PCR followed, using primers 3557/3558. The same method was used to construct knockout strains of the E3 ligase-related genes in the ISW1K97R strain. Briefly, the up- or downstream genomic DNA sequences of the target genes were amplified using the primers listed in the Supplementary Data primer table.

The ISW1-FLAG complementation strains were generated as described henceforth. The downstream genomic DNA sequence of ISW1 was amplified using primers 3735/3736 and cloned between the restriction sites SacII and SacI into the pFlag-NAT plasmid (a plasmid containing the nourseothricin-resistance marker and the Flag tag). The complete ORF of the target gene (including the promoter sequence) was amplified using primers 3733/3734 and cloned between the restriction sites HindIII and EcoRI into pFlag-NAT-dw. The cassette amplified from the final pFlag-NAT by primer 3898-MY/3899-MY was biolistically transformed into the isw1∆::NEO strain. Diagnostic PCR was performed using the 3733/3734 primer pair. Real-time PCR analysis was performed using a gene-specific probe amplified using the 3557/4421-MY primer pair. Western blot analysis of Isw1 was performed using anti-Flag mouse monoclonal antibodies.

The R mutation was formed using site-directed mutagenesis approaches. The K89 codon was first mutated using primers 3733/3866-MY and 3891/3734, then the K89R construct was amplified using the 3733/3734 primer pair and cloned between the restriction sites HindIII and EcoRI into pFlag-NAT-dw. The K97 codon was mutated using primers 3733/3787 and 3862-MY/3734, whereas the K113 codon was mutated using primers 3733/3836 and 3864-MY/3734. The resulting plasmids contained single-point mutants (K89R, K97R, or K113R), double-site mutants (K89R, K97R; K89R, K113R; or K97R, K113R) and triple-site mutant (K89R, K97R, and K113R). The Q mutation was formed using the same procedures except that the K89Q construct was formed using the primer pair 3733/3866-MY and 3892/3734, K97Q was formed using the primer pair 3733/3787 and 3863-MY/3734 and K113Q was formed using the primer pair 3733/3836 and 3865-MY/3734. The resulting plasmid contained single-point mutants (K89Q, K97Q, or K113Q), double-site mutants (K89Q, K97Q; K89Q, K113Q; or K97Q, K113Q), and triple-site mutant (K89Q, K97Q, and K113Q). All mutant plasmids were further confirmed using DNA sequencing. The cassette amplified from the final pFlag-NAT by primer 3898-MY/3899-MY was biolistically transformed into the isw1∆::NEO strain. Mutant strains were confirmed using DNA sequencing, diagnostic PCR, qRT-PCR, and immunoblotting.

Ubiquitination mutants were also formed. The mutant K147R was formed using primer pair 3733/4445-MY and 4444-MY/3734, whereas K183R was amplified using the primer pair 3733/4447-MY and 4446-MY/3734 and K297R was mutated using primers 4448-MY/4449-MY. Similarly, K347R was mutated using primers 4450-MY/4451-MY, K415R was mutated using primers 3807-MY/3809-MY and K441R was mutated using primers 4452-MY/4453-MY. The resulting plasmids were used to generate the R mutant plasmid using the TaKaRaMutanBEST Kit (Takara). All strains were validated using the methods described earlier.

To demonstrate the direct protein interaction between Isw1 and Cdc4, the downstream genomic DNA sequence of CDC4 was amplified using primers ZR34/ZR51 and cloned between the restriction sites SpeI and SacI into the pHA-HYG plasmid (a plasmid containing the hygromycin B-resistance marker and the HA tag). Then, the last 1000 bp of the CDC4 ORF was amplified using primers ZR48 and ZR33, and the resulting fragment was cloned into the above plasmid between ClaI and SmaI. The cassette amplified from the final plasmid by primers ZR48 and ZR51 was biolistically transformed into H99, the ISW1WT, ISW1K97Q, and ISW1K97R strains. Diagnostic PCR was performed using the ZR48/ZR51 primer pair. Immunoblotting analysis of Cdc4 was performed using anti-HA (C29F4) rabbit mAb. The following strains were constructed using the same experimental procedure: Itc1-HA/Isw1-Flag, Chd1-HA/Isw1-Flag, and Sua7-HA/Isw1-Flag.

To detect the protein expression levels of Isw1 in clinical strains, the wild-type plasmid with pFlag-NAT was used as a template in PCR using primers 3557 and 3537. The resulting PCR products were transformed into seven clinical strains. The ISW1K97Q overexpression strains were generated as described henceforth. A safe-haven site was applied to perform plasmid integration (Arras et al., 2015). The 3′ flanking region of the safe haven was amplified using 4470-MY/4471-MY, then cloned into pFlag-NAT between the SacII and SacI sites. The 5′ flanking region of the safe haven was amplified using primer 4800-MY/4467-MY, the TEF1 promoter was amplified using primer 4468-MY/2342 and the ISW1K97Q coding sequence was amplified using 4807-MY and 3736. The ISW1K97Q construct was amplified using the 4800-MY/3736 primer pair, and the three gel-extracted DNA fragments from the first and second rounds of PCR were used as templates, then cloned into pFlag-NAT-dw between the HindIII and EcoRI sites. The ISW1K97Q overexpression cassette was amplified using the 4800-MY/4471-MY primer pair, and the product was transformed into clinical strains.

Animal infection and in vivo drug-resistance tests

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Mice were anesthetized and inoculated intranasally with 105 yeast cells suspended in 50 μl PBS buffer. Infected mice were weighed 12 days after infection and then monitored twice daily for morbidity. Mice were sacrificed at the endpoint of the experiment. All animal experimentation was carried out under the approved protocol (please see Ethical statement).

The experimental design involved the implementation of in vivo investigations using a pre-established intranasal infection model as outlined by Oliveira NK (Oliveira et al., 2021). Each treatment group and control group consisted of 10 female BALB/c mice. Fungal cells were incubated for a period of 24 hr in a 10 ml volume of YPD medium at a temperature of 30°C. Subsequently, the cells were subjected to two rounds of washing and subsequently resuspended in PBS. All mice were subjected to an infection by intranasally introducing 105 yeast cells. At 24 hr after infection, the mice were administered a single treatment with either FLC (at doses of 45 or 5 mg/kg) or 5-FC (at doses of 200 or 100 mg/kg) through intraperitoneal injection. The control group received a saline solution (PBS). The therapies were administered on a daily basis for a duration of 7 consecutive days. On the 14th day following infection, lung tissues were removed and homogenized, and then CFU analyses were performed.

Protein co-IP mass spectrometry

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Mid-log phase cells from ISW1-FLAG complementation strains subcultured at 30°C in 50 ml YPD medium were employed. Lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% NP-40; pH 7.5) with 1× protease inhibitor cocktail (CWBIO) and 40 mM PMSF removed cell proteins. All lysate protein samples were treated overnight with Sigma anti-Flag magnetic beads at 4°C. After three TBS buffer washes, the bound proteins were extracted into protein loading buffer (125 mM Tris–HCl, 4% SDS, 20% glycerol; pH 7.5) at 95°C for 5 min. The protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness for digestion. Add 100 mM triethylammonium bicarbonate buffer (TEAB) to urea below 2 M to dilute the protein sample. Finally, trypsin was added at 1:50 for the first overnight digestion and 1:100 for the second 4 hr digestion. Finally, C18 SPE column desalted peptides.

The tryptic peptides were diluted in solvent A (0.1% formic acid, 2% acetonitrile/water) and put onto a homemade reversed-phase analytical column (25 cm length, 75/100 μm i.d.). A nanoElute UHPLC system (Bruker Daltonics) separated peptides with a gradient from 6% to 24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24% to 35% in 14 min, 80% in 3 min, and 80% for 3 min at 450 nl/min. Capillary source and timsTOF Pro (Bruker Daltonics) mass spectrometry were used on the peptides. The electrospray voltage was 1.60 kV. The TOF detector evaluated precursors and fragments with a 100–1700 m/z MS/MS scan range. PASEF mode was used on the timsTOF Pro. Precursors having charge states 0–5 were selected for fragmentation, and 10 PASEFMS/MS scans were obtained per cycle. The dynamic exclusion was 30 s.

MaxQuant search engine (v.1.6.15.0) handled MS/MS data. Tandem mass spectra were searched against the reverse decoy database and C. neoformans Protein-Fungi database (7429 entries). Up to two missed cleavages were allowed with trypsin/P. In first search, precursor ions had a mass tolerance of 20 ppm, in main search, 5 ppm, and fragment ions 0.02 Da. FDR was lowered to <1%.

All differentially expressed protein database accession or sequence was searched for protein–protein interactions in STRING 11.5. Only interactions between proteins in the searched dataset were chosen, avoiding extraneous candidates. STRING uses a ‘confidence score’ metric to measure interaction confidence. We identified interactions with a confidence value ≥0.15 (low confidence).

Transcriptome and qRT-PCR analyses

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To analyze drug resistance, the wild-type H99 and isw1Δ mutant strains were either untreated or were treated with 10 μg/ml FLC (Sigma) in 50 ml YPD media at 30°C until cell densities reached the exponential phase (approximately 6–7 hr). Cells were then washed three times with ice-cold PBS and placed in a tank of liquid nitrogen. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific), and 3 μg of the product was processed using the TruSeq RNA Sample Preparation Kit (Illumina). Purification of mRNA was performed using polyT oligo-attached magnetic beads. Fragmentation of mRNA was performed using an Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using random hexamer primers and SuperScript II. Subsequently, second-strand cDNA was synthesized using RNase H and DNA polymerase I. The 3′ end of the cDNA sequence was adenylated, then cDNA sequences of 200 bp were purified using the AMPure XP system (Beckman Coulter) and enriched using an Illumina PCR Primer Cocktail in a 15-cycle PCR. The resulting PCR products were then purified, and integrity was confirmed using an Agilent High Sensitivity DNA assay on a Bioanalyzer 2100 (Agilent). The sequencing library was then sequenced using a Hiseq platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd. Alignments were checked against the Cryptococcus_neoformans_var._grubii_H99 reference genome and gene annotation set retrieved from Ensemble. Differentially expressed genes were detected using the Bioconductor package DESeq2 version 1.22.2 (Gao et al., 2022; Love et al., 2014). Genes with adjusted p-values <0.05 and changes greater or less than 1.5-fold those of the control strain were considered to be significantly induced or repressed, respectively.

To verify the gene changes screened by transcriptomics, H99 and isw1Δ mutant strains were either untreated or were treated with 40 μg/ml FLC (MedChemExpress) in 10 ml YPD media at 30°C, and cell densities were monitored until OD600 reached 1.0. Both the H99 and isw1Δ mutant strains were either untreated or treated with 400 μg/ml 5-FC (MedChemExpress) in 10 ml YPD media at 30°C for 1 hr. Cells were harvested at 3000 rpm for 3 min at 4°C, then washed twice with ice-cold PBS. Total RNA was isolated using a total RNA kit I (Omega), and cDNA was synthesized using a reverse transcript all-in-one mix (Mona). Primers for amplifying target genes can be found in the primer table. Data were acquired using a CFX96 real-time system (Bio-Rad) using actin expression as a normalization control. The ΔΔCt method was used to calculate differences in expression.

Co-IP and immunoblotting assays

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Overnight cultures of C. neoformans strains were diluted in fresh YPD media and incubated at the indicated temperature to the mid-log phase (OD600 = 0.8). Protein immunoprecipitation or co-IP was performed as described elsewhere (Li et al., 2019). Briefly, cell proteins were extracted using lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% NP-40; pH 7.5) with 1× protease inhibitor cocktail (CWBIO) and 40 mM PMSF. Aliquots of protein extracts were retained as input samples. Samples of the lysed protein were incubated with anti-Flag magnetic beads (MedChemExpress) at 4°C overnight, then the beads were washed three times using TBS buffer, and the bound proteins were extracted into protein loading buffer at 95°C for 5 min. Protein samples were separated using 8% SDS–PAGE, transferred onto nitrocellulose membranes, and blocked using 5% milk. Immunoblotting or co-IP assays were performed using anti-Flag mouse monoclonal antibodies (1:5000 dilution; Transgene), anti-HA (C29F4) rabbit mAb (1:5000 dilution; Cell Signaling Technology), anti-Histone H3 (D1H2) XP Rabbit mAb (1:5000 dilution; Cell Signaling Technology), goat anti-mouse IgG (H+L) HRP secondary antibodies, and goat anti-rabbit IgG (H+L) HRP secondary antibodies (1:5000 dilution; Thermo Fisher Scientific), and monoclonal and polyclonal Kac (1:2500; PTM Bio). The mouse anti-acetyllysine primary antibody (clone Kac-10; PTM Bio, Cat No. PTM-101) was used to detect Kac (Li et al., 2019; Xu et al., 2023). The signal was captured using a ChemiDoc XRS+ (Bio-Rad). The resulting pictures were analyzed and quantified using Image Lab version 5.2.

Statistical analysis

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All statistical analyses were performed using GraphPad Prism software (GraphPad 6.0). Two-tailed unpaired t-tests were used in two-sample comparisons. Statistical analyses for two or more groups were performed using one- or two-way analysis of variance. Significant changes were recognized when p < 0.05. All experiments were performed using at least three biological replicates to ensure reproducibility.

Detection of drug content

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To analyze drug resistance, the wild-type H99 and isw1Δ mutant strains were treated with 40 μg/ml FLC in 50 ml YPD media at 30°C until the cell densities reached the exponential phase (approximately 5 hr), then the cells were washed once with PBS. An appropriate amount of uniform sample was weighed, 0.2 ml 50% acetic acid solution was added, ultrasonic extraction was carried out, and the resultant was passed through a 0.22-μm microporous filter membrane. High-performance liquid chromatography was performed using an injection volume of 10 nl and a constant mobile phase flow rate of 1.0 ml/min. An Agilent C18 (4.6 mm × 250 mm × 5 μm) column was used, held at 35°C, on a Thermo U3000 HPLC; the detector was a DAD. When the drug was FLC, the mobile phase was acetonitrile:water:acetic acid (25:75:0.2), the detector wavelength was 261 nm, the run time was 15 min, and the standard curve was Y = 0.0147X − 0.0109 (r2 = 0.9999).

The drug content was determined as:

W=(CC0)VNm

W—drug content, mg/kg

C—concentration of the drug in the cell, mg/l

C0—concentration of the drug in the blank control, mg/l

V—volume, ml

N—diluted

m—cell mass, g

Data availability

The raw Isw1 proteome modification mass spectrometric data have been deposited to the Proteome Xchange with identifier PXD037150. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository (Chen et al., 2022) with the dataset identifier PXD045338. The transcriptomics data (RNA-seq) is deposited in NCBI's Gene Expression Omnibus (GEO) and can be accessed through GEO Series accession ID GEO:GSE217187 and GSE235148. Any other data necessary to support the conclusions of this study are available in the supplementary data files and source data. Reagents and fungal strains are available from the authors upon request.

The following data sets were generated
    1. Ding C
    (2022) NCBI Gene Expression Omnibus
    ID GSE217187. Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans.
    1. Yang M
    (2022) PRIDE
    ID PXD037150. Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans.
    1. Yang M
    (2023) ProteomeXchange
    ID PXD045338. Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans.
    1. Ding C
    (2023) NCBI Gene Expression Omnibus
    ID GSE235148. Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans.

References

    1. Arras SD
    2. Chitty JL
    3. Blake KL
    4. Schulz BL
    5. Fraser JA
    (2015)
    A genomic safe haven for mutant complementation in Cryptococcus neoformans
    PLOS ONE 10:e0122916.
    1. Erwig LP
    2. Gow NA
    (2016)
    Interactions of fungal pathogens with phagocytes
    Nat Rev Microbiol 14:163–176.
  1. Report
    1. World Health Organization
    (2022)
    WHO fungal priority pathogens list to guide research, development and public health action
    World Health Organization.

Decision letter

  1. Detlef Weigel
    Senior and Reviewing Editor; Max Planck Institute for Biology Tübingen, Germany

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Interplay between acetylation and ubiquitination of imitation switch chromatin Cryptococcus neoformans" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Arturo Casadevall as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

We would welcome a revision that addresses all the reviewer points below. In addition please pay particular attention to these comments that emerged during the reviewer discussion of your paper.

1) it is important to more firmly demonstrate whether the Isw1 effect is due to its chromatin activity.

2) Isw1 is not well characterised in C. neoformans. It is important to demonstrate that Isw1-protein complexes are conserved between different organisms- the authors should have this info thanks to their Mass-Spec data.

3) Can you explain how Iswi was found to be important in antifungal resistance? Are Iswi paralogs present in Cryptococcus? What potential Iswi interacting proteins are present in Cryptococcus other than Itc1?

4) Can you explain the lack of high molecular-weight ubiquitinated Isw1 proteins (I am not an expert in ubiquitination but I thought that poly-ubiquitination always produced a different ladder) and

5) The specificity of the KAc antibody used needs to be better defined.

6) The authors should calculate the MIC using standard methods.

The revised manuscript will be sent back to the reviewers for re-review so please make sure you answer all the points above and below if you resubmit to eLife.

Reviewer #1 (Recommendations for the authors):

1. It would greatly enhance the paper if the authors could show that the isw1 mutant is resistant to antifungal treatment in mouse models. Given that the mutant is fully virulent, the difference in survival or fungal burden after drug treatment would indicate the potential role of Isw1 during antifungal therapy. That would enhance the impact of the research showing the potential clinical relevance of Isw1 regulation.

2. The MICs shown in Figure 1b and Figure 6f were not done according to standard protocols. Although the spotting assays are nice because they are visual, the authors need to use the accepted standard protocol to determine MICs of antifungals for both the mutants and the clinical isolates.

3. It's surprising that all the clinical isolates used in this study showed altered Isw1 levels. The authors could include clinical isolates with known mechanisms of resistance for comparison.

4. The authors believe that reduction in 5-FC uptake contributes to the resistance of the isw1 mutant to this drug. Given that isw1 mutation altered the expression of many genes (but none of the changes of the transporter genes are huge as the log2 FPKM is within 1.5, Figure 1C) and that there is only one known transporter Fcy2 involved in the intake of 5-FC, this presents an ideal case for the authors to demonstrate that reduced uptake is indeed the underlying resistance mechanism.

Reviewer #2 (Recommendations for the authors):

General comments:

The term protein posttranslational modification (PPTM) is more commonly referred to as post-translational modification (PTM). The authors use PPTM throughout this manuscript. The authors could consider changing "PPTM" to "PTM".

The authors' use of "increase", "induce", or similar wording is confusing in some instances. For example, in Lines 197-198 the authors suggest that deacetylase inhibitors "increase acetylation levels" (see also Lines 235, 283, and 284 for additional examples). The authors should consider rewording these examples and reviewing the manuscript for similar examples to clarify their results.

The authors find that deletion of ITC1, which is known to interact with Isw1 homologs in other systems, results in increased antifungal resistance. However, loss of Itc1 does not fully phenocopy loss of Isw1 (see Figure 1—figure supplement 2 panel B). This is likely due to the activity of other Isw1 interacting proteins. The authors are encouraged to review recent literature on other organisms (particularly other fungi, such as Neurospora crassa) on other potential interacting proteins.

The authors found that deletion of CDC4 in the Isw1 K97R background results in reduced resistance to antifungals. It also appears that the deletion of CDC4 in the Isw1 K97R background impacts general growth as shown on YPD in Figure 5 (panel A). Did the authors examine the deletion of CDC4 in the wild-type background? How does deletion of CDC4 in wild type impact general growth and resistance to antifungals?

The authors state that "results showed an interaction between Cdc4 and Isw1 K97R but

not between Cdc4 and Isw1 K97Q" (Line 269) in reference to data shown in Figure 5 (panel E). However, it appears that Cdc4 and Isw1 K97Q show a very weak interaction based on the co-IP data. The authors should address this discrepancy.

The authors are encouraged to thoroughly review and revise the "Material and Methods" section and figure legends of the manuscript to ensure methods are described in sufficient detail to repeat, citations are provided when needed, and figures are adequately described. For example: (1) although the manuscript relies heavily on the quantification of western blot data the authors do not describe how this quantification was performed, and (2) the authors do not describe how RNA-seq data was processed or mapped (see Line 559) or provide a citation for DESeq2 package (see Line 562).

It is not clear the number of biological replicates used or if experiments were repeated for some experiments. For example: (1) in Figure 3 panels A and B and related source data, it appears these experiments were run once with one biological replicate, and (2) in Figure 3 panel F and related source data, it appears that western blots for two replicates are shown, although the data for three replicates are plotted. Could the authors clarify the number of replicates used and if experiments were repeated?

Specific comments:

Line 68: The authors should rewrite/edit Line 68 for clarity.

Line 69: The citation, "(2017)", is incomplete. The authors should correct the citation here and check throughout the manuscript for additional instances of incomplete citations (e.g., Line 74).

Line 103: It appears the Florent et al., 2009 citation is incorrectly used here, as the referenced study investigated antifungal resistance in Candida lusitaniae.

Line 134: The authors could consider briefly describing why they chose to study Isw1 in Cryptococcus.

Line 136: The authors should specify that the isw1 mutant was complemented with an ISW1 allele tagged with FLAG.

Line 154: The authors state "To test if this increase affects drug uptake,…"; however, the experiment described doesn't distinguish between decreased drug import and increased drug export. This should be rewritten for clarity.

Line 180: The authors should rewrite/edit Line 180 for clarity.

Line 191: The authors should rewrite/edit Line 191 for clarity.

Line 200: The authors should rewrite/edit Line 200 for clarity and define the abbreviation for DNA binding domain as only "DBD" is used in Figure 2 (panel F) to avoid confusion. Additionally, the authors should describe why certain acetylated residues were chosen and others were not.

Lines 201-202: The authors should revise the statement "to mimic a fully acetylated Isw1" for clarity and correctness, as there appear to be additional acetylated lysine residues in Isw1.

Lines 208-209: The authors should rewrite/edit Lines 208-209 for clarity.

Lines 220-221: The authors should rewrite/edit Lines 220- a221 for clarity.

Line 246: Data presented in this study suggest 15 residues of Isw1 are ubiquitinated but only six were mutated. The authors should provide reasoning for selecting certain ubiquitination sites in the manuscript. Specifically, it is curious that the authors did not test K98 as the proximity to K97 could affect acetylation or vice versa. Can the authors explain why K98 was not tested? Additionally, the authors state that "five failed to affect drug-resistant growth phenotypes". However, it seems that two of the six tested residues had an impact on drug resistance in the K97R background. The authors should address this discrepancy.

Lines 250-252: The authors should rewrite/edit Lines 250-252 for clarity.

Line 277: "majority was" should be changed to "majority were" in this instance.

Lines 307-308: Gcn5 is not a deacetylase but is the catalytic subunit of the histone acetyltransferase complexes, ADA, and SAGA. The authors should address this discrepancy.

Line 313: The authors use "the acetylation-Isw1-ubiquitination axis" here and use the "Isw1 acetylation-ubiquitin-proteasome regulation axis" above (see Line 289). The authors could consider using consistent wording throughout the manuscript for clarity.

Line 315: It appears the authors use a lowercase "L" instead of an uppercase "I" for Ioc3 and Ioc4. The authors should address this discrepancy.

Lines 320-323: The authors do not directly demonstrate that transporter-encoding genes upregulated in the ISW1 deletion strain are responsible for reduced FLC in cells or if the reduction of FLC in cells is caused by decreased drug import and/or increased drug export (see comment for Line 154 above). Relatedly, it should be noted that Figure 1 (panel C) indicates multiple transporter-encoding genes are downregulated in the ISW1 deletion strain, and it is not clear if the authors describe and/or discuss this in the text. With these points in mind, the authors should rewrite Lines 320-323 for clarity.

Lines 324-327: The authors should rewrite/edit Lines 324-327 for correctness and clarity. For example, the authors state "Isw1 is a transcription activator for genes responsible for resistance to FLC"; however, it seems that genes involved in FLC resistance are upregulated in the absence of Isw1, which means Isw1 activity represses expression of such genes. The authors are also encouraged to keep in mind that Isw1 is an ATP-dependent chromatin remodeling factor and not a transcription factor.

Figure 2 panels B and D: The authors should define CHX in the figure legend, i.e., "cycloheximide (CHX)".

Figure 4 panel E: It appears the images showing growth on 16 ug/ml FLC and 20 ug/ml FLC may be mislabeled. The authors should review and correct if needed.

Reviewer #3 (Recommendations for the authors):

A) How did the author identify ISWI as a candidate gene important for regulating drug resistance in Cryptococcus?

B) The budding yeast, S. cerevisiae, expresses two homologs of ISWI, Isw1p, and Isw2p. Isw2p associates with Itc1p (Sugiyama and Nikawa, 2001) The Isw1p forms two distinct complexes: one subcomplex is formed by Isw1p, Ioc2p, and Ioc4p and a second by Isw1p and Ioc3 (Vary Mol Cell Biol 2003). Does Cryptococcus contain two ISWI genes? If so, why did the authors focus just on the Isw(2?)-Itc1p proteins? Does Cryptococcus encode for Ioc2, Ioc3, and Ioc4? The authors should clarify this point.

C) The itc1 deletion strain is barely resistant to FLC (Figure S2): MIC for isw1 δ and itc1 δ strains should be calculated.

D) Did the authors perform transcriptomic analysis of WT and isw1 deleted cells without FLC? The material and methods section suggests that this experiment has been performed (Pg 18 line 544). However, these RNA-seq data are not shown in Figure 1. This is a critical experiment as it establishes whether ISWI is an activator or a repressor of gene expression.

E) Isw1-FLAG pulldown- It is strange that the authors dive into Isw1 post-translational modifications without describing the pull-down and characterising the protein complexes. What is the efficiency of the pull-down? This could be shown by Silver staining+ Western blot. What are the Iswi1-interacting proteins? Are the ISWI-protein complexes conserved in Cryptococcus?

F) For all the FLAG-Western Blots: No FLAG control is missing.

G) For all the FLAG Pulldowns: Input is missing.

H) What does the anti-Kac detect? Iswi1 acetylation? Did the authors raise the antibody? Histone acetylation? I cannot find this info in the Material and Methods. Assuming that the antibody detects Isw1 acetylation, I am not convinced that "the presence of antifungal agents strongly repressed acetylation levels" (Line 196) as reduced protein acetylation levels are observed only when FLC treatment is combined with TSA/NAM treatment.

I) Although the mutation analysis demonstrates that K97 plays a role in the drug response- data demonstrating that acetylation of this residue regulates this process are missing. Could the mutation affect ISWI folding? The crystal structure of several ISWI proteins has been solved and it should be relatively easy to predict the effect of K97 mutations on ISWI protein folding.

J) Protein poly-ubiquitination (normally linked to proteosome-degradation) leads to higher molecular weight protein species in a Western blot analysis. Therefore, it is puzzling that these high molecular weight species are not detected, especially after MG132 treatment.

K) Is the Cdc4-Iswi interaction detected by Mass Spec Analysis?

L) Is ISWI function in drug resistance linked to its chromatin remodelling activity?

M) Does ISWI interact with chromatin? If so, which are ISWI-target genes? Does drug treatment modulate chromatin binding?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans" for further consideration by eLife. Your revised article has been evaluated by Detlef Weigel (Senior Editor) and a Reviewing Editor.

While the manuscript has been improved there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

General comments on revision: The revised manuscript is much improved overall. The authors have performed numerous additional experiments that strengthen the study. The majority of my initial comments have been addressed, and I thank the authors for carefully considering the comments and their responses. However, there are a few key points in the manuscript (largely associated with revisions or new data) that should be addressed to further strengthen the study and broaden its impact.

Prior reviewer comment: The authors demonstrate that Isw1 has a role in responding to antifungals in Cryptococcus. However, it is not clear if changes in Isw1 stability represent a general response to stress. This study would have benefited from experiments to test: (1) if levels of Isw1 change in response to other stressors (e.g., heat, osmotic, or oxidative stress) and (2) if loss of Isw1 impacts resistance to other stressors.

Author response: A series of experiments were conducted to illustrate and measure phenotypic traits associated with virulence. These traits encompassed capsule formation, melanin synthesis, cell proliferation under stressful conditions, and Isw1 expression levels in response to diverse environmental stimuli. Please see Figure 3a, 3b, 3c, Figure 3—figure supplement 1 and line 237-241.

Reviewer response: The authors provide new data to address the initial comment. The phenotypic data presented in Figure 3a, 3b, and 3c are convincing, although it should be noted that there appears to be a slight reduction in melanin production in Figure 3b. The data presented in Figure 3 —figure supplement 1 is less convincing. It is likely difficult to say "that Isw1 expression is not affected by external stress inducers" (line 237) considering data presented in Figure 1 —figure supplement 1 and Figure 4. Specifically, (1) ISW1 expression is not affected by antifungal treatment under the conditions tested and (2) changes in Isw1 levels associated with antifungal treatment are observed after inhibiting protein synthesis. It is thus plausible that changes in Isw1 levels under stress conditions could be missed in the assay presented in Figure 3 —figure supplement 1. The authors should change the text accordingly.

Prior reviewer comment: The authors find that deletion of ITC1, which is known to interact with Isw1 homologs in other systems, results in increased antifungal resistance. However, loss of Itc1 does not fully phenocopy loss of Isw1 (see Figure 1 —figure supplement 2 panel B). This is likely due to the activity of other Isw1 interacting proteins. The authors are encouraged to review recent literature on other organisms (particularly other fungi, such as Neurospora crassa) on other potential interacting proteins.

Author response: We acknowledge the significance of the conservation of Isw1 across many species. The regulatory pathway of Isw1 was initially discovered in the model organism Saccharomyces cerevisiae, which possesses two paralogs, namely Isw1 and Isw2, as a result of a whole genome duplication event(Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997). Due to the absence of a complete genome duplication event in C. neoformans, its genome solely contains a single copy of the ISW gene. Prior research conducted on Saccharomyces cerevisiae has provided evidence that the ISWI complex is composed of several subunits, namely Isw1, Ioc proteins, Itc1, Chd1, and Sua7 (Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013). The genome of C. neoformans was examined, and it was observed that the IOC gene family could not be identified. This suggests that the IOC gene family has likely undergone an evolutionary loss in C. neoformans, as indicated on the FungiDB website. The genome of C. neoformans harbors three distinct genes, namely Itc1, Chd1, and Sua7. In order to comprehensively investigate the cryptoccocal ISWI complex, we conducted a methodical Isw1-Flag protein immunoprecipitation procedure, which was subsequently followed by Mass-Spec analysis. In the present study, a total of 22 proteins that interacted with Isw1 were discovered. Among these proteins, 11 have been previously reported to be associated with the regulatory networks including Isw1. In the mass spectrometry results, Itc1 was found to be co-immunoprecipitated with Isw1. Although the Mass-Spec analysis did not reveal the presence of Chd1 and Sua7, our study demonstrated that Chd1 can be coimmunoprecipitated with Isw1 through co-IP and immunoblotting techniques. However, no interaction between Isw1 and Sua7 was established utilizing any of these methods. In order to gain a deeper understanding of the involvement of Chd1 and Itc1 in the regulation mechanism of Isw1 in multidrug resistance, we created strains with disrupted chd1Δ and itc1Δ genes. The only strain that exhibited a drug resistance phenotype similar to that of the isw1Δ strain was the itc1Δ strain. The data presented in this study indicate that there has been evolutionary divergence in the Isw1-protein complexes between the species C. neoformans and S. cerevisiae. The manuscript has undergone significant modifications. Please see Figure 2 and text line 208-232.

Reviewer response: The authors provide new data to data address the initial comment. This data greatly improves the manuscript. However, it also raises multiple concerns that must be addressed.

1. The authors should be mindful that (1) there is not a single "ISWI Complex" in S. cerevisiae or other eukaryotes, but rather ISWI homologs form multiple complexes (e.g., Isw1a, Isw1b, and Isw2 in S. cerevisiae); and (2) the IOC (Iswi One Complex) genes encoding Ioc2, Ioc3, and Ioc4 are not a "gene family" per se as the proteins are quite different, and it may be difficult to readily identify divergent homologs in C. neoformans. Additionally, the authors should clarify what is meant by "canonical ISWI complex" (line 231).

2. It is unclear why the authors chose to focus on Chd1 and Sua7 as (1) neither Chd1 nor Sua7 appear in the AP-MS data and (2) there is very little evidence that Chd1 or Sua7 directly interact with Isw1 or Isw2 in S. cerevisiae (to the best of my knowledge, Michaelis et al., 2023 Nature provides the only biochemical evidence for interaction between Isw1 and Chd1 in S. cerevisiae). There is evidence that both Chd1 and Sua7 genetically interact with Isw1 or Isw2 in S. cerevisiae, which is what is described in the references cited by the authors (for example, Smolle et al., 2012). Along these lines, the authors should review the text and references and revise both accordingly. It should be noted that the evidence for an association between Isw1 and Chd1 in C. neoformans is quite interesting (although it may not be a direct physical interaction).

3. As mentioned in the initial comment, the authors are encouraged to review recent literature on ISWI homologs in organisms other than S. cerevisiae (e.g., Neurospora crassa – Kamei et al., 2021 PNAS and Wiles et al., 2022 eLife) to strengthen this aspect of the manuscript. For example, there are at least two additional proteins in the AP-MS data that are well-characterized to interact with ISWI homologs. Along these lines, the authors should provide protein names in addition to locus IDs in Figure 2a (as was done for Itc1) to help readers and broaden interest.

Prior reviewer comment: The authors' use of "increase", "induce", or similar wording is confusing in some instances. For example, in Lines 197-198 the authors suggest that deacetylase inhibitors "increase acetylation levels" (see also Lines 235, 283, and 284 for additional examples). The authors should consider rewording these examples and reviewing the manuscript for similar examples to clarify their results.

Author response: The manuscript has been appropriately updated in accordance with the given instructions.

Reviewer response: Many such instances were not corrected (for example, see lines 281 and 322-323). Similarly, (1) in lines 127-128, the authors state "Cryptococcal Isw1 plays an indispensable role in modulating drug-resistance genes", but the role of Isw1 is not indispensable since the isw1 mutant strain has increased resistance to antifungals; and (2) in line 247, the authors state "that the isw1 mutant strain exhibits improved fungal burdens", but the isw1 mutant strain showed increased fungal burdens not improved fungal burdens.

https://doi.org/10.7554/eLife.85728.sa1

Author response

Essential revisions:

We would welcome a revision that addresses all the reviewer points below. In addition please pay particular attention to these comments that emerged during the reviewer discussion of your paper.

1) it is important to more firmly demonstrate whether the Isw1 effect is due to its chromatin activity.

In order to investigate the potential role of Isw1 on chromatin activity in the modulation of multidrug resistance, we have conducted protein truncation experiments. Specifically, we deleted the DNA binding domain, the helicase domain, and the SNF2 domain, which have been previously shown to regulate Isw1 chromatin activity in the model organism S. cerevisiae (Grune T, 2003; Mellor J, 2004; Pinskaya M, 2009; Rowbotham SP, 2011). The new data demonstrated that all truncation variants of Isw1 mutants had a growth phenotype consistent with that of the deletional strain isw1Δ. In addition, the levels of gene expression observed in these strains were also similar to those observed in the deletion strain isw1Δ. This finding provides evidence that the regulation of the drug resistance mechanism is influenced by these critical domains involved in modifying chromatin activities. Moreover, the Isw1-Flag strain was utilized to conduct chromatin immunoprecipitation and PCR experiments, which revealed that Isw-1 exhibits the ability to directly bind to the promoter regions of target genes. The new findings added evidence substantially supporting the hypothesis that the Isw1 chromatin activity plays a crucial role in modulating its protein function and acting as a central regulator of drug resistance in C. neoformans.

Please see revised Figure 1g, 1h, 1i, and lines 186-199 in the revised manuscript text.

2) Isw1 is not well characterised in C. neoformans. It is important to demonstrate that Isw1-protein complexes are conserved between different organisms- the authors should have this info thanks to their Mass-Spec data.

We acknowledge the significant concern raised over the conservation of Isw1 across several species. The regulatory mechanism of Isw1 was initially discovered in S. cerevisiae. This process involves two paralogs, Isw1 and Isw2, which emerged as a result of the complete genome duplication event ((Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997)). Because no evidence suggested that C. neoformans has gone through similar genome duplication, only one copy of ISW gene was identified. Previous research in S. cerevisiae has provided evidence that the ISWI complex is comprised of several subunits, namely Isw1, Ioc, Itc1, Chd1, and Sua7 (Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013). Upon a thorough examination of the C. neoformans genome, we have not been able to identify a similar IOC gene family. This absence likely suggests an evolutionary loss of the IOC gene family in C. neoformans, as reported on the FungiDB website (https://fungidb.org/fungidb/app). However, C. neoformans has Itc1, Chd1, and Sua7. While we concur with the aforementioned statement on the capability of Mass-Spec data to elucidate potential protein-protein interactions and aid in the identification of subunits within the ISWI complex, it is important to acknowledge that the PTM Mass-Spec methodology is solely employed for the purpose of identifying potential sites of protein modification. In order to comprehensively investigate the cryptoccocal ISWI complex, we conducted a standardized Isw1-Flag protein immunoprecipitation procedure, followed by Mass-Spec analysis. In the present study, a total of 22 proteins that interacted with Isw1 were found. Among these proteins, 11 have been previously reported to be associated with the regulatory networks including Isw1. In the mass spectrometry results, Itc1 was found to be co-immunoprecipitated with Isw1. Although the Mass-Spec analysis did not reveal the presence of Chd1 and Sua7, our study demonstrated that Chd1 can be coimmunoprecipitated with Isw1 through co-IP and immunoblotting techniques. However, no interaction between Isw1 and Sua7 was established utilizing any of these methods. In order to gain a deeper understanding of the involvement of Chd1 and Itc1 in the regulation mechanism of Isw1 in multidrug resistance, we created chd1Δ and itc1Δ disruption mutant strains. The only strain that exhibited a drug resistance phenotype similar to the isw1Δ strain is the itc1Δ strain. The new data indicated that there is evolutionary divergence in the Isw1-protein complexes between C. neoformans and S. cerevisiae. We have added new findings and revised relevant statements. Please see new Figure 2, Supplementary File 4, and text lines 206–232.

3) Can you explain how Iswi was found to be important in antifungal resistance? Are Iswi paralogs present in Cryptococcus? What potential Iswi interacting proteins are present in Cryptococcus other than Itc1?

Isw1 was identified in a further investigation built upon the findings presented in our previously published studies (Li Y, 2019). In this study, the acetylome in C. neoformans was comprehensively analyzed, and a series of knockout strains were created to investigate the relationship between acetylation and fungal pathogenicity. The isw1 mutant was discovered to be a modifier of drug resistance. The identification of fungal paralogs of ISW genes was initially observed in S. cerevisiae, which has two paralogs, Isw1 and Isw2, as a result of genome duplication (Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997). In contrast, C. neoformans has only one ISW1 gene. We have conducted bioinformatic analysis, protein co-immunoprecipitation followed by Mass Spectrometry, immunoblotting experiments, and gene disruption studies to characterize Isw1. In addition, we found that C. neoformans does not have all of the IOC proteins as S. cerevisiae does and the cryptococcal Isw1-protein associated complex exhibits substantial divergence from the yeast. We established connections between Isw1-Itc1 and -Chd1, but not Isw1-Sua7, in C. neoformans. Consequently, we have conducted additional investigations to also elucidate the role of Chd1 and Itc1 in regulating drug resistance. It is noteworthy that among the studied strains, only Itc1 exhibited a reciprocal regulation of drug resistance in C. neoformans. This particular phenotype bears resemblance to that seen in the cryptoccocal isw1Δ strain.

Please see revised Figure 2, Supplementary File 4, and text lines 206–232.

4) Can you explain the lack of high molecular-weight ubiquitinated Isw1 proteins (I am not an expert in ubiquitination but I thought that poly-ubiquitination always produced a different ladder) and

In theory, the process of poly-ubiquitination results in the formation of high molecular-weight proteins that exhibit smear patterns. However, when employing the immunoblotting technique to analyze poly-ubiquitination proteins, it is common to observe the detection of the apo form of the target proteins (i.e., non-ubiquitinated forms of the protein). This is mostly due to the low abundance of ubiquitinated protein species. It is also crucial to note that the presence of a smear in a detection assay does not provide definitive evidence of ubiquitination, as the smear may be attributed to various other factors. Hence, the identification of ubiquitinated proteins necessitates an additional enrichment step, such as the protein immunoprecipitation (IP) assay, followed by the detection of ubiquitin through the utilization of anti-ubiquitin antibodies. In addition, it should be noted that mass spectrometry is considered the most precise technique for the detection of ubiquitination. It is worth mentioning that our original manuscript included data obtained using MS analysis. In order to provide a more precise account of conventional techniques employed for the detection of ubiquitination using immunoblotting, we presented below two illustrative instances of ubiquitination detection investigations that have been recently published. Both experimental protocols involved IP, followed by immunoblotting analysis employing anti-ubiquitin (UB) antibodies.

Study 1. Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis. 2019, Nature, https://doi.org/10.1038/s41586-020-2928-y

Please see Figure 1 for HMGCR protein IP followed by Ub detection using the Ub antibody

Study 2. USP10 strikes down b-catenin by dual-wielding deubiquitinase activity and phase separation potential, Cell Chemical Biology. https://doi.org/10.1016/j.chembiol.2023.07.016

Please see Figure E,F,H for Axin1 protein IP followed by Ub detection using the Ub antibody

In our studied, we have seen that the native form of Isw1 can be detected using immunoblotting, but the presence of high molecular weight protein species remains undetectable. This finding aligns with the outcomes reported in the aforementioned papers. In the revised manuscript, an attempt was made to investigate the impact of IP Isw1 and measure ubiquitination levels using commercially available mammalian Ub antibodies. However, these efforts did not yield successful results. In order to address the concerns raised, we successfully engineered a strain of C. neoformans capable of producing the human UBB1 protein. Experiments using ubiquitination were conducted. Three strains were subjected to testing, namely the wild-type strain, the ISW1K97Q strain, and the ISW1K97R strain. The cells were subjected to incubation in media that were either supplemented with MG132 or not. The experimental protocol involved protein pull down utilizing Flag beads, followed by the identification and quantification of ubiquitin with anti-human ubiquitin antibodies. Following an extended period of exposure, we successfully observed the presence of poly-ubiquitinated protein smear bands, which corresponded to the elevated molecular weight of poly-ubiquitinated Isw1. The results revealed that the presence of poly-ubiquitinated Isw1 species exclusively in the ISW1K97R strain, while not in the wild-type and ISW1K97Q strains. The presence of the K97Q mutant inhibits the ubiquitination of Isw1. These new data provided additional evidence to support the hypothesis that Isw1 is a protein undergoing ubiquitination. Furthermore, our findings suggested that the acetylation site at position 97 plays a crucial role in regulating the ubiquitination process, acting as a switch to turn it on or off.

We have updated our results and statements.

5) The specificity of the KAc antibody used needs to be better defined.

We regret for not included the antibody information in the prior version of the manuscript. The pan Kac antibody is a specific Kac antibody that has been widely employed in numerous acetylation studies (Please refer to the following two recent articles that have utilized the pan antibody:). The antibody information has been updated in the modified manuscript. Please see line 837-838.

1. Li, et al. Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence[J].Communications Biology.2019

2. Liu, et al. SIRT7 couples light-driven body temperature cues to hepatic circadian phase coherence and gluconeogenesis[J].Nature Metabolism.2019

6) The authors should calculate the MIC using standard methods.

We highly value these crucial suggestions. In the revised manuscript, we have conducted the drug inhibitory experiments again, employing the previously established technique. Additional quantitative results have been provided. Please see Figures 1b, 2e, 8e-8i, and line 562-580.

The revised manuscript will be sent back to the reviewers for re-review so please make sure you answer all the points above and below if you resubmit to eLife.

Reviewer #1 (Recommendations for the authors):

1. It would greatly enhance the paper if the authors could show that the isw1 mutant is resistant to antifungal treatment in mouse models. Given that the mutant is fully virulent, the difference in survival or fungal burden after drug treatment would indicate the potential role of Isw1 during antifungal therapy. That would enhance the impact of the research showing the potential clinical relevance of Isw1 regulation.

We express our gratitude for your significant and insightful feedback. We concur with the reviewer's assertion that understanding the control of Isw1 in the context of systemic infection is of utmost importance in terms of its potential clinical significance. In the revised manuscript, a series of experiments were conducted to validate the in vivo drug resistance of Isw1 regulation. Initially, we conducted experiments to ascertain that Isw1 does not function as a virulence regulator. This was achieved by evaluating the levels of capsule formation, melanin synthesis, and cell proliferation in response to adverse environmental conditions. The data presented in this study indicate that the protein levels of Isw1 are not subject to regulation by environmental pressures. Additionally, no growth abnormalities were observed in the isw1 mutant strain under these conditions. Next, we conducted an experiment to assess the involvement of the isw1 mutant strain in fungal colonization in mice. The findings revealed that the fungal load of the isw1 mutant strain was similar to that of the wildtype strain. Hence, the isw1 mutant exhibits suitability for subsequent analysis. Subsequently, mice were subjected to infection with either wildtype or mutant fungal cells, followed by the administration of chemotherapeutic agents. The data presented in our study revealed a significant increase in fungal load in the isw1 mutant cells as compared to the wildtype cells following the administration of 5-FC or FLC in mice. Hence, the updated results provide substantial evidence to support the notion that Isw1 plays a pivotal role in the regulation of drug resistance, both in vitro and in vivo organisms. Please see Figure 3 and line 234-250.

2. The MICs shown in Figure 1b and Figure 6f were not done according to standard protocols. Although the spotting assays are nice because they are visual, the authors need to use the accepted standard protocol to determine MICs of antifungals for both the mutants and the clinical isolates.

We appreciate this essential comments. In the revised manuscript, we have redone all the drug inhibitory tests using protocol described previously. More quantitative results have been updated. Please see Figures 1b, 2e, 8e-8i, and line 562-580.

3. It's surprising that all the clinical isolates used in this study showed altered Isw1 levels. The authors could include clinical isolates with known mechanisms of resistance for comparison.

We express our genuine gratitude for this comment. Firstly, the development of drug resistance in fungi is a complex mechanism that encompasses various regulators, including mutations in crucial gene sets and regulatory pathways. In addition, fungal cells employ distinct mechanisms of resistance to effectively counteract various categories of antifungal drugs. For instance, fungal cells engage different processes to develop resistance to 5-FC and FLC. Hence, the inclusion of one or more resistance players may significantly introduce additional variables, thereby impeding the study. Moreover, considering the extensive repertoire of known resistance mechanisms in fungi, the analysis of Isw1's function would be further complicated. Furthermore, this research, conducted in collaboration with local hospitals, has successfully found multiple strains of C. neoformans that exhibit resistance (as shown in Supplementary File 1 sheet2). However, it is worth noting that these strains do not possess any recognized mechanisms of resistance. In other words, the sequencing analysis conducted on these strains did not identify any mutations in genes known to be associated with drug resistance (see to Supplementary File 5 for more details). Although our analysis did not include any clinical strains with known mechanisms of resistance, it has effectively illustrated a significant association between Isw1 and drug resistance in the laboratory standard strain, H99. Moreover, through the manipulation of Isw1 protein levels in the clinical strains, we have demonstrated in great detail that the concentration of Isw1 protein has the ability to impact the susceptibility of cells to antifungal drugs. In response to this worry, our group has undertaken the initiative of expanding our clinical collections over a period of six months. The regulation of Isw1 was examined in a total of twelve clinical isolates. In this study, Isw1-Flag constructs were produced in an additional six clinical isolates, namely CDLC4, CDLC6, CDLC37, CDC43, CDLC100, and CDLC141. The protein expression data demonstrated the presence of three separate categories. 1. Strains exhibiting traits of multidrug resistance demonstrated significantly reduced levels of Isw1-Flag. The strains encompassed in this set are CDLC15, CDLC25, CDLC61, CDLC62, and CDLC98. 2. On the other hand, strains that demonstrated susceptibility to drugs revealed substantial protein expression levels of Isw1-Flag. Prominent instances of these strains include CDCL120, CDCLC6, CDCL37, CDCLC43, and CDLC100. Two clinical isolates that displayed resistance to antifungal medications, yet had high quantities of the Isw1 protein. Subsequent examination of these strains revealed that the levels of Isw1 exert regulatory influence over drug resistance in those strains. In the context of the CDCLC141 study, it is seen that despite the presence of a higher level of Isw1, the expression of a stable form of Isw1 actually diminishes drug resistance. Hence, the Isw1 regulatory axis might be considered a prototypical clinical phenomena. Please see figure 8, supplementary file 5 and line 373–416.

4. The authors believe that reduction in 5-FC uptake contributes to the resistance of the isw1 mutant to this drug. Given that isw1 mutation altered the expression of many genes (but none of the changes of the transporter genes are huge as the log2 FPKM is within 1.5, Figure 1C) and that there is only one known transporter Fcy2 involved in the intake of 5-FC, this presents an ideal case for the authors to demonstrate that reduced uptake is indeed the underlying resistance mechanism.

We much value the insightful feedback provided by the reviewer. However, it is our contention that this expert may have misconstrued our intracellular quantification results for FLC. In both the prior and revised manuscript, we included a quantitative analysis of intracellular fluconazole concentration, but not of 5-FC. Initially, we observed an upregulation in the expression levels of genes responsible for encoding drug pumps. Specifically, we identified a total of 12 pumps, consisting of 7 members from the ABC family, 2 efflux pumps, and 3 pumps from the MSF family. In order to conduct a more accurate assessment of the control of Isw1, it is advisable to create a strain whereby the genes responsible for the functioning of these 12 pumps are disrupted, within the background of an isw1 deletion strain. Nevertheless, due to the extensive number of genes implicated and the potential variations in pump combinations that may contribute to the resistance mechanism, the suggested method is deemed unsuitable for procurement. Alternatively, we inquired about the potential involvement of Isw1 in the modulation of drug efflux. In this regard, it may be feasible to assess the intracellular concentration of FLC as a means to more accurately ascertain the drug tolerance state of the isw1 mutant strain. No recognized receptors for fluconazole (FLC) have been found in fungal organisms, and drug efflux pumps have been directly associated with the development of drug tolerance. Hence, from the quantification of intracellular FLC concentration and the investigation of pump gene expression, it is highly probable that Isw1 plays a role in regulating drug resistance by modifying drug efflux. In both the prior and current iterations of the work, we have not conducted quantitative analysis of intracellular 5-FC concentration. This is due to the fact that 5-FC functions as a prodrug, undergoing rapid metabolism and conversion into several downstream molecules. Consequently, the quantification of 5-FC becomes more challenging. However, a substantial body of prior literature has comprehensively illustrated the association between 5-FC and resistance genes, including Fcy2, Fcy1, and other significant genes. Significantly, Isw1 not only governs the entry of drugs (Fcy2), but it also modulates both canonical and non-canonical mechanisms of 5-FC drug resistance pathway, encompassing four pivotal genes. Consequently, we have utilized a comprehensive integration of biochemical, genetic, and molecular biological experimental evidence, along with corroborating published findings, to demonstrate that Isw1 exerts control over FLC resistance by regulating the expression of drug pumps, and governs 5-FC resistance by modulating the expression of 5-FC metabolic pathways. The manuscript has been revised. Please see line 145-153 and 167-178.

Reviewer #2 (Recommendations for the authors):

General comments:

The term protein posttranslational modification (PPTM) is more commonly referred to as post-translational modification (PTM). The authors use PPTM throughout this manuscript. The authors could consider changing "PPTM" to "PTM".

All PPTMs have been changed to PTMs.

The authors' use of "increase", "induce", or similar wording is confusing in some instances. For example, in Lines 197-198 the authors suggest that deacetylase inhibitors "increase acetylation levels" (see also Lines 235, 283, and 284 for additional examples). The authors should consider rewording these examples and reviewing the manuscript for similar examples to clarify their results.

The manuscript has been appropriately updated in accordance with the given instructions.

The authors find that deletion of ITC1, which is known to interact with Isw1 homologs in other systems, results in increased antifungal resistance. However, loss of Itc1 does not fully phenocopy loss of Isw1 (see Figure 1—figure supplement 2 panel B). This is likely due to the activity of other Isw1 interacting proteins. The authors are encouraged to review recent literature on other organisms (particularly other fungi, such as Neurospora crassa) on other potential interacting proteins.

We acknowledge the significance of the conservation of Isw1 across many species. The regulatory pathway of Isw1 was initially discovered in the model organism Saccharomyces cerevisiae, which possesses two paralogs, namely Isw1 and Isw2, as a result of a whole genome duplication event(Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997). Due to the absence of a complete genome duplication event in C. neoformans, its genome solely contains a single copy of the ISW gene. Prior research conducted on Saccharomyces cerevisiae has provided evidence that the ISWI complex is composed of several subunits, namely Isw1, Ioc proteins, Itc1, Chd1, and Sua7 (Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013). The genome of C. neoformans was examined, and it was observed that the IOC gene family could not be identified. This suggests that the IOC gene family has likely undergone an evolutionary loss in C. neoformans, as indicated on the FungiDB website. The genome of C. neoformans harbors three distinct genes, namely Itc1, Chd1, and Sua7. In order to comprehensively investigate the cryptoccocal ISWI complex, we conducted a methodical Isw1-Flag protein immunoprecipitation procedure, which was subsequently followed by Mass-Spec analysis. In the present study, a total of 22 proteins that interacted with Isw1 were discovered. Among these proteins, 11 have been previously reported to be associated with the regulatory networks including Isw1. In the mass spectrometry results, Itc1 was found to be co-immunoprecipitated with Isw1. Although the Mass-Spec analysis did not reveal the presence of Chd1 and Sua7, our study demonstrated that Chd1 can be coimmunoprecipitated with Isw1 through co-IP and immunoblotting techniques. However, no interaction between Isw1 and Sua7 was established utilizing any of these methods. In order to gain a deeper understanding of the involvement of Chd1 and Itc1 in the regulation mechanism of Isw1 in multidrug resistance, we created strains with disrupted chd1Δ and itc1Δ genes. The only strain that exhibited a drug resistance phenotype similar to that of the isw1Δ strain was the itc1Δ strain. The data presented in this study indicate that there has been evolutionary divergence in the Isw1-protein complexes between the species C. neoformans and S. cerevisiae. The manuscript has undergone significant modifications. Please see Figure 2 and text line 208-232.

The authors found that deletion of CDC4 in the Isw1 K97R background results in reduced resistance to antifungals. It also appears that the deletion of CDC4 in the Isw1 K97R background impacts general growth as shown on YPD in Figure 5 (panel A). Did the authors examine the deletion of CDC4 in the wild-type background? How does deletion of CDC4 in wild type impact general growth and resistance to antifungals?

We express our gratitude for this comment. In the revised publication, we created a cdc4Δ deletion strain and subsequently shown that the absence of cdc4Δ does not exert any discernible impact on drug resistance. It is postulated that the observed phenomenon may be attributed to the presence of other E3 ligases that modulate the stability of the Isw1 protein. This speculation arises from the observation that, even in the absence of Cdc4 in the K97R mutant strain, a certain degree of drug resistance growth is still exhibited. The manuscript has been revised to incorporate this information. Please see Figure 7—figure supplement 1b, and line 356.

The authors state that "results showed an interaction between Cdc4 and Isw1 K97R but

not between Cdc4 and Isw1 K97Q" (Line 269) in reference to data shown in Figure 5 (panel E). However, it appears that Cdc4 and Isw1 K97Q show a very weak interaction based on the co-IP data. The authors should address this discrepancy.

We concur with the assertion that the K97Q mutation resulted in a reduction in the strength of the interaction between Isw1 and Cdc4, rather than a complete blockade. The manuscript has been revised in accordance with the suggested revisions. Please see line 362-367.

The authors are encouraged to thoroughly review and revise the "Material and Methods" section and figure legends of the manuscript to ensure methods are described in sufficient detail to repeat, citations are provided when needed, and figures are adequately described. For example: (1) although the manuscript relies heavily on the quantification of western blot data the authors do not describe how this quantification was performed, and (2) the authors do not describe how RNA-seq data was processed or mapped (see Line 559) or provide a citation for DESeq2 package (see Line 562).

We have included all necessary details and references in the Material and Methods. Please see line 820-824 and line 858. (Gao X, 2022; Love MI, 2014).).

It is not clear the number of biological replicates used or if experiments were repeated for some experiments. For example: (1) in Figure 3 panels A and B and related source data, it appears these experiments were run once with one biological replicate, and (2) in Figure 3 panel F and related source data, it appears that western blots for two replicates are shown, although the data for three replicates are plotted. Could the authors clarify the number of replicates used and if experiments were repeated?

We apologize for not providing the necessary replicate information. We have modified the text, and including the raw data for biological replicates in the source data. Please see line 1360 and source data figure 5 source data 8.

Specific comments:

Line 68: The authors should rewrite/edit Line 68 for clarity.

Please see line 51-53.

Line 69: The citation, "(2017)", is incomplete. The authors should correct the citation here and check throughout the manuscript for additional instances of incomplete citations (e.g., Line 74).

Please see line 53, 60.

Line 103: It appears the Florent et al., 2009 citation is incorrectly used here, as the referenced study investigated antifungal resistance in Candida lusitaniae.

Please see line 89

Line 134: The authors could consider briefly describing why they chose to study Isw1 in Cryptococcus.

Please see line 128–134.

Line 136: The authors should specify that the isw1 mutant was complemented with an ISW1 allele tagged with FLAG.

Please see line 136.

Line 154: The authors state "To test if this increase affects drug uptake,…"; however, the experiment described doesn't distinguish between decreased drug import and increased drug export. This should be rewritten for clarity.

Please see line 162–166.

Line 180: The authors should rewrite/edit Line 180 for clarity.

Please see line 260-262.

Line 191: The authors should rewrite/edit Line 191 for clarity.

Please see line 273.

Line 200: The authors should rewrite/edit Line 200 for clarity and define the abbreviation for DNA binding domain as only "DBD" is used in Figure 2 (panel F) to avoid confusion. Additionally, the authors should describe why certain acetylated residues were chosen and others were not.

Please see Figure 1i and line 197–201.

Lines 201-202: The authors should revise the statement "to mimic a fully acetylated Isw1" for clarity and correctness, as there appear to be additional acetylated lysine residues in Isw1.

Please see line 282-284.

Lines 208-209: The authors should rewrite/edit Lines 208-209 for clarity.

Please see line 291-293.

Lines 220-221: The authors should rewrite/edit Lines 220- a221 for clarity.

Please see line 305–306.

Line 246: Data presented in this study suggest 15 residues of Isw1 are ubiquitinated but only six were mutated. The authors should provide reasoning for selecting certain ubiquitination sites in the manuscript. Specifically, it is curious that the authors did not test K98 as the proximity to K97 could affect acetylation or vice versa. Can the authors explain why K98 was not tested? Additionally, the authors state that "five failed to affect drug-resistant growth phenotypes". However, it seems that two of the six tested residues had an impact on drug resistance in the K97R background. The authors should address this discrepancy.

In light of the above criticism, we have undertaken further measures to induce a mutation in the K98 site, resulting in the development of the R variant within the genetic background of the K97 mutation strain. The findings of our study indicate that modifications to the K98 sites do not have any discernible effect on drug resistance. In the present study, our research aimed to investigate the interplay between acetylation and ubiquitination in the modulation of drug resistance. In order to mitigate the substantial workload associated with simultaneously altering all 15 ubiquitination sites, our research team made the decision to concentrate our efforts on the sites neighboring the K97 site. This strategic approach was based on the logical assumption that these adjacent sites possess the greatest potential for being influenced by the K97 sites. There is a possibility that additional ubiquitination sites may play a significant role in regulating the stability of the Isw1 protein. The study's findings indicate the presence of reciprocal control between acetylation and ubiquitination. The current data obtained in our research substantiates this principle and offers compelling evidence in support of this conclusion. The manuscript has been revised to provide a description of the process involved in selecting Ub locations. Please see line 333-340.

Lines 250-252: The authors should rewrite/edit Lines 250-252 for clarity.

Please see line 341-344.

Line 277: "majority was" should be changed to "majority were" in this instance.

Please see line 395–404.

Lines 307-308: Gcn5 is not a deacetylase but is the catalytic subunit of the histone acetyltransferase complexes, ADA, and SAGA. The authors should address this discrepancy.

Please see line 434-436.

Line 313: The authors use "the acetylation-Isw1-ubiquitination axis" here and use the "Isw1 acetylation-ubiquitin-proteasome regulation axis" above (see Line 289). The authors could consider using consistent wording throughout the manuscript for clarity.

We have modified text. Please see line 440.

Line 315: It appears the authors use a lowercase "L" instead of an uppercase "I" for Ioc3 and Ioc4. The authors should address this discrepancy.

We have modified text. Please see line 442.

Lines 320-323: The authors do not directly demonstrate that transporter-encoding genes upregulated in the ISW1 deletion strain are responsible for reduced FLC in cells or if the reduction of FLC in cells is caused by decreased drug import and/or increased drug export (see comment for Line 154 above). Relatedly, it should be noted that Figure 1 (panel C) indicates multiple transporter-encoding genes are downregulated in the ISW1 deletion strain, and it is not clear if the authors describe and/or discuss this in the text. With these points in mind, the authors should rewrite Lines 320-323 for clarity.

We have modified the text. Please see line 455–459.

Lines 324-327: The authors should rewrite/edit Lines 324-327 for correctness and clarity. For example, the authors state "Isw1 is a transcription activator for genes responsible for resistance to FLC"; however, it seems that genes involved in FLC resistance are upregulated in the absence of Isw1, which means Isw1 activity represses expression of such genes. The authors are also encouraged to keep in mind that Isw1 is an ATP-dependent chromatin remodeling factor and not a transcription factor.

We have modified this. Please see line 464-468.

Figure 2 panels B and D: The authors should define CHX in the figure legend, i.e., "cycloheximide (CHX)".

We have modified this. Please see line 1258 and 1264.

Figure 4 panel E: It appears the images showing growth on 16 ug/ml FLC and 20 ug/ml FLC may be mislabeled. The authors should review and correct if needed.

We have modified this. Please see Figure 6e.

Reviewer #3 (Recommendations for the authors):

A) How did the author identify ISWI as a candidate gene important for regulating drug resistance in Cryptococcus?

We express our gratitude for this criticism. The identification of Isw1 was conducted as a subsequent study to our previously published data (Li Y, 2019). In prior research, we conducted a comprehensive analysis of the acetylome in C. neoformans, and then developed knockout strains to investigate the relationship between acetylation and fungal pathogenicity. The Isw1 mutant has been discovered as a modifier of drug resistance. Please see line 129-134.

B) The budding yeast, S. cerevisiae, expresses two homologs of ISWI, Isw1p, and Isw2p. Isw2p associates with Itc1p (Sugiyama and Nikawa, 2001) The Isw1p forms two distinct complexes: one subcomplex is formed by Isw1p, Ioc2p, and Ioc4p and a second by Isw1p and Ioc3 (Vary Mol Cell Biol 2003). Does Cryptococcus contain two ISWI genes? If so, why did the authors focus just on the Isw(2?)-Itc1p proteins? Does Cryptococcus encode for Ioc2, Ioc3, and Ioc4? The authors should clarify this point.

We acknowledge the significant concern raised over the conservation of Isw1 across several species. The regulatory mechanism of Isw1 was initially discovered in the model organism Saccharomyces cerevisiae. This process involves two paralogs, Isw1 and Isw2, which emerged as a result of the complete genome duplication event (Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997). Because C. neoformans has not gone through the complete genome duplication event, its genome only encodes one copy of ISW gene. Prior research conducted on Saccharomyces cerevisiae has provided evidence that the ISWI complex is comprised of several subunits, namely Isw1, Ioc genes, Itc1, Chd1, and Sua7 (Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013). Upon doing a thorough examination of the genome of C. neoformans, our investigation yielded negative results in terms of identifying the IOC gene family. This absence likely suggests an evolutionary loss of the IOC gene family in C. neoformans, as suggested on the FungalDB website. The genome of C. neoformans has the genes Itc1, Chd1, and Sua7. While we concur with the aforementioned statement on the capability of Mass-Spec data to elucidate potential protein-protein interactions and aid in the identification of subunits within the ISWI complex, it is important to acknowledge that the PTM Mass-Spec methodology is solely employed for the purpose of identifying potential sites of protein modification. In order to comprehensively investigate the cryptoccocal ISWI complex, we conducted a standardized Isw1-Flag protein immunoprecipitation procedure, followed by Mass-Spec analysis. In the present study, a total of 22 proteins that interact with Isw1 were found in our experimental data. Among these proteins, 11 have been previously reported to be associated with the regulatory networks including Isw1. In the mass spectrometry results, the protein Itc1 was found to be co-immunoprecipitated with the protein Isw1. Although the Mass-Spec analysis did not reveal the presence of Chd1 and Sua7, our study demonstrated that Chd1 can be coimmunoprecipitated with Isw1 through the utilization of co-IP and immunoblotting techniques. However, no interaction between Isw1 and Sua7 was shown utilizing any of these methods. The data presented in this study indicate that there has been evolutionary divergence in the Isw1-protein complexes between C. neoformans and S. cerevisiae. The manuscript has undergone significant modifications. Please see Figure 2 and line 206-232.

C) The itc1 deletion strain is barely resistant to FLC (Figure S2): MIC for isw1 δ and itc1 δ strains should be calculated.

We highly value these crucial remarks. In the revised manuscript, we have conducted the drug inhibitory tests once again, following the previously demonstrated technique (Xie J et al., 2012). Additional quantitative results have been provided. Please refer to Figures 2e and Line 224-228. The strain with the deletion of itc1 gene exhibits resistance to antifungal drugs.

D) Did the authors perform transcriptomic analysis of WT and isw1 deleted cells without FLC? The material and methods section suggests that this experiment has been performed (Pg 18 line 544). However, these RNA-seq data are not shown in Figure 1. This is a critical experiment as it establishes whether ISWI is an activator or a repressor of gene expression.

In the revised manuscript, we conducted a transcriptome analysis of both wildtype and isw1 deletion strains in the absence of FLC treatment. When comparing the data collected under two different settings, namely with and without FLC treatment, we observed a distinct difference in the regulation of gene expression between these two conditions. In the case of the isw1 deletion strain subjected to FLC treatment, a total of 21 genes, encompassing the ABC/MFS family and efflux pumps, exhibited substantial alterations in gene expression. Specifically, 9 genes were downregulated while 12 genes were upregulated. Conversely, when FLC supplementation was not provided, only 9 genes demonstrated changes in gene expression, with 3 genes being downregulated and 6 genes being upregulated. Hence, the Isw1 protein is essential for the activation of specific genes, while concurrently exerting a repressive role on other genes. The manuscript has been revised to accurately and comprehensively depict our findings. Please see Figure 1c, Supplementary File 2 and line 145-153.

E) Isw1-FLAG pulldown- It is strange that the authors dive into Isw1 post-translational modifications without describing the pull-down and characterising the protein complexes. What is the efficiency of the pull-down? This could be shown by Silver staining+ Western blot. What are the Iswi1-interacting proteins? Are the ISWI-protein complexes conserved in Cryptococcus?

We express our gratitude for this comment. Pull-down studies are frequently employed as a prevalent method for analyzing post-translational modifications (PTMs) of a particular protein in cases where antibodies specific to PTM sites are not accessible. The used methodology involves the utilization of protein pull-down technique in conjunction with pan antibody detection for the purpose of acetylation analysis. This approach aims to enhance the concentration of the target protein by employing commercially available beads specifically designed for epitope tag isolation. In our investigation, Flag beads (Σ) were utilized for this purpose. Subsequently, specialized antibodies, such as a pan anti-lysine acetylation antibody, are employed to ascertain the overall amounts of acetylation in Isw1. In order to investigate the protein complexes associated with Isw1 in the organism C. neoformans, our study employed a series of scientific methodologies including bioinformatic analysis, protein co-immunoprecipitation followed by Mass Spectrometry, immunoblotting experiments, and gene disruption analyses. Prior research conducted on Saccharomyces cerevisiae has identified several proteins that interact with Isw1, namely Ioc proteins, Itc1, Sua7, and Chd1(Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013).. However, it has been observed that the regulatory network of the cryptococcal Isw1-protein associated complex exhibits substantial divergence when compared to that of S. cerevisiae. The absence of the IOC gene family in the genome of C. neoformans indicates a distinct regulatory pattern of Isw1 between the two species. Furthermore, the detection of protein interaction between Isw1 and Sua7 in S. cerevisiae has not been seen through the utilization of Mass-Spec and Co-IP assays in C. neoformans. The connections between Isw1-Itc1 and -Chd1 were indeed confirmed in C. neoformans, indicating a certain degree of preservation of the ISWI complex machinery throughout fungal evolution. Subsequently, we have undertaken additional efforts to elucidate the role of Chd1 and Itc1 in the regulation of the multidrug resistance mechanism. It is noteworthy that among the tested strains, only Itc1 exhibited a reciprocal regulation of drug resistance in C. neoformans. This particular phenotype bears resemblance to that seen in the cryptoccocal isw1Δ strain. In summary, the ISWI regulatory mechanism of cryptococcal organisms exhibits a distant relationship to that found in S. cerevisiae. Please see Figure 2 and line 206-232.

F) For all the FLAG-Western Blots: No FLAG control is missing.

In this investigation, we utilized the Flag antibody produced by Σ, a well-established manufacturer that has been widely exploited in previous research, leading to a substantial amount of publications. The Σ antibody does not generate any non-specific bands when tested against both the wildtype strain (H99) and the FLAG strains. In order to provide further clarification regarding the specific antibody, our study incorporates a series of studies comprising eight biological duplicates and three sets of complete blots obtained from three different exposure durations. The presence of Isw1 was observed at around 130 kDa, while no discernible band corresponding to the H99 strain was found. Hence, this antibody yields outcomes that are both specific and dependable.

Author response image 1

G) For all the FLAG Pulldowns: Input is missing.

In the manuscript, all inputs were provided as depicted in Figure 5a and 5b. This comment suggests that the observed acetylation results are obtained without any inputs. The regular input samples are utilized to investigate the presence of proteins of interest. In the context of PTM analysis, the detection method involves the utilization of a pan anti-acetyllysine antibody for the purpose of PTM detection. Routine inputs can only generate a broad representation of the overall acetyllysine levels throughout the cell. However, these input samples cannot serve as a control group for accurately quantifying specific target proteins or post-translational modifications (PTMs). Please refer to the figure provided below, which displays the prior publications as well as the input samples utilized for the detection of acetylation.

Author response image 2

H) What does the anti-Kac detect? Iswi1 acetylation? Did the authors raise the antibody? Histone acetylation? I cannot find this info in the Material and Methods. Assuming that the antibody detects Isw1 acetylation, I am not convinced that "the presence of antifungal agents strongly repressed acetylation levels" (Line 196) as reduced protein acetylation levels are observed only when FLC treatment is combined with TSA/NAM treatment.

We regret the omission of the antibody information in the prior iteration of the manuscript. The pan Kac antibody is a highly specific antibody targeting lysine acetylation (Kac), which has been extensively employed in numerous studies involving acetylation analysis. Please refer to the provided references for further information. The revised manuscript includes updated information regarding the antibodies. The statement has been updated. Please see line 836-838.

1. Qiutao Xu, et al. ROS-stimulated Protein Lysine Acetylation Is Required for Crown Root Development in Rice[J].Journal of Advanced Research.2022.

2. Nan Liu, et al. HDAC inhibitors improve CRISPR/Cas9 mediated prime editing and base editing[J].Molecular Therapy-Nucleic Acids.2022.

3. Yi Fang, et al. Histone crotonylation promotes mesoendodermal commitment of human embryonic stem cells[J].Cell Stem Cell.2021.

4. Li, et al. Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence[J].Communications Biology.2019.

5. Shun Tu, et al. YcgC represents a new protein deacetylase family in prokaryotes[J].eLife.2015.

Author response image 3

I) Although the mutation analysis demonstrates that K97 plays a role in the drug response- data demonstrating that acetylation of this residue regulates this process are missing. Could the mutation affect ISWI folding? The crystal structure of several ISWI proteins has been solved and it should be relatively easy to predict the effect of K97 mutations on ISWI protein folding.

In light of the aforementioned comment, a comprehensive structural algorithm analysis was conducted on both Isw1 wildtype and mutant variants. The results of the prediction analysis did not reveal any substantial changes in the folding of Isw1.

J) Protein poly-ubiquitination (normally linked to proteosome-degradation) leads to higher molecular weight protein species in a Western blot analysis. Therefore, it is puzzling that these high molecular weight species are not detected, especially after MG132 treatment.

We express our gratitude for this comment. In theory, the process of poly-ubiquitination results in the formation of high molecular-weight proteins that exhibit smear patterns. However, when employing the immunoblotting technique to analyze poly-ubiquitination proteins, it is common to observe the detection of the apo form of the target proteins (i.e., non-ubiquitinated forms of the protein). This is mostly due to the low abundance of ubiquitinated protein species. It is also crucial to note that the presence of a smear in a detection assay does not provide definitive evidence of ubiquitination, as the smear may be attributed to various other factors. Hence, the identification of ubiquitinated proteins necessitates an additional enrichment step, such as protein immunoprecipitation assay, followed by the detection of ubiquitin through the utilization of anti-ubiquitin antibodies. In addition, it should be noted that mass spectrometry is considered the most precise technique for the detection of ubiquitination. It is worth mentioning that the manuscript in its prior iteration included data obtained using MS analysis. In order to provide a more precise account of conventional techniques employed for the detection of ubiquitination using immunoblotting, we present below two illustrative instances of ubiquitination detection investigations that have been recently published. Both experimental protocols involved the utilization of the protein immunoprecipitation (IP) technique, followed by immunoblotting analysis employing anti-ubiquitin (UB) antibodies.

Study 1.Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis. 2019, Nature, https://doi.org/10.1038/s41586-020-2928-y

Please see Figure 1 for HMGCR protein IP followed by Ub detection using Ub antibody

Study 2.USP10 strikes down b-catenin by dual-wielding deubiquitinase activity and phase separation potential, Cell Chemical Biology. https://doi.org/10.1016/j.chembiol.2023.07.016

Please see Figure E,F,H for Axin1 protein IP followed by Ub detection using Ub antibody

In our investigation, we have seen that the native form of Isw1 can be detected using immunoblotting, but the presence of high molecular weight protein species remains undetectable. This finding aligns with the outcomes reported in the aforementioned papers. In the revised manuscript, an attempt was made to investigate the impact of IP Isw1 and afterwards measure ubiquitination levels using commercially available mammalian Ub antibodies. However, these efforts did not yield successful results. In order to address the concerns raised in this statement, we successfully engineered a strain of C. neoformans capable of producing the human UBB1 protein. Experiments using ubiquitination were conducted. Three strains were subjected to testing, namely the wildtype strain, the ISW1K97Q strain, and the ISW1K97R strain. The cells were subjected to incubation in medium that were either supplemented with MG132 or lacking MG132. The experimental protocol involved conducting protein pull down experiments utilizing Flag beads, followed by the subsequent identification and quantification of ubiquitin through the utilization of anti-human ubiquitin antibodies. Following an extended period of exposure, we successfully observed the presence of poly-ubiquitinated protein smear bands, which corresponded to the elevated molecular weight of poly-ubiquitinated Isw1. The results reveals that the presence of poly-ubiquitinated Isw1 species is observed exclusively in the ISW1K97R strain, while no such species are detected in the wildtype and ISW1K97Q strains. The presence of the K97Q mutant inhibits the ubiquitination of Isw1, so providing additional evidence to support the hypothesis that Isw1 is a protein that undergoes ubiquitination. Furthermore, our findings suggest that the acetylation site at position 97 plays a crucial role in regulating the ubiquitination process, acting as a switch to turn it on or off.

K) Is the Cdc4-Iswi interaction detected by Mass Spec Analysis?

In the revised manuscript, we conducted Isw1-Flag pull-down experiments followed by Mass spectrometry analysis. However, our findings did not reveal the presence of Cdc4. This can be attributed mostly to the limitations of the mass spectrometry technique, which resulted in insufficient coverage of the proteome. Despite recent advancements in proteomic technologies, the task of achieving a thorough coverage of an organism or sample's proteome remains tough. Certain proteins have the potential to evade detection or pose challenges in their identification, hence impeding a comprehensive comprehension of the entirety of the protein landscape. Nevertheless, our experimental approach including protein co-immunoprecipitation (co-IP) and subsequent immunoblotting provided evidence of the interaction between Cdc4 and Isw1. Please see Table S4.

L) Is ISWI function in drug resistance linked to its chromatin remodelling activity?

In order to investigate the potential role of Isw1 chromatin activity in the modulation of multidrug resistance, we conducted protein truncation experiments. Specifically, we genetically deleted the DNA binding domain, helicase domain, and SNF2 domain, which have been previously shown to regulate Isw1 chromatin activity in the model organism S. cerevisiae (Grune T, 2003; Mellor J, 2004; Pinskaya M, 2009; Rowbotham SP, 2011). The data demonstrates that all shortened variants of Isw1 mutants had a growth phenotype characterized by multidrug resistance, which is consistent with the growth phenotype observed in the deletion strain isw1Δ. Furthermore, the levels of gene expression observed in these strains were found to be similar to those observed in the deletion strain isw1Δ. This finding provides evidence that the regulation of the drug resistance mechanism is actually influenced by these critical domains involved in modifying chromatin activity. In addition, the Isw1-Flag strain was utilized to conduct chromatin immunoprecipitation and PCR experiments, which revealed that Isw-1 exhibits the ability to directly bind to the promoter regions of target genes. The findings from this collective analysis of the revised data provide substantial evidence supporting the notion that the Isw1 chromatin activity plays a crucial role in modulating its protein function, acting as a central regulator of drug resistance in C. neoformans. Please see Figure 1g, 1h, 1i and line 186-199.

M) Does ISWI interact with chromatin? If so, which are ISWI-target genes? Does drug treatment modulate chromatin binding?

To effectively tackle this concern, we have pursued two distinct approaches to demonstrate the chromatin regulatory effects of Isw1. In this study, the DNA binding domain was deliberately removed through genetic manipulation. The data presented indicates that the Isw1 mutants with shorter variations exhibited a growth phenotype that was characterized by multidrug resistance. This growth phenotype correlates with the growth phenotype obtained in the isw1Δ deletion strain. Additionally, it was observed that the levels of gene expression in the strain were comparable to those detected in the deletion strain isw1Δ. This discovery offers empirical support for the notion that the control of the drug resistance mechanism is indeed impacted by the DNA binding capability of Isw1. Furthermore, the Isw1-Flag strain was employed to perform chromatin immunoprecipitation and PCR assays, demonstrating the direct binding capacity of Isw1 to the promoter regions of target genes. The results obtained from this comprehensive analysis of the revised data offer significant evidence for the proposition that Isw1 interacts with chromatin and that its chromatin activity plays a pivotal role in modulating its protein function. This interaction serves as a central regulatory mechanism for drug resistance in C. neoformans. Furthermore, a transcriptome analysis was performed on both wildtype and isw1 deletion strains in the absence of FLC therapy. Upon comparing the results obtained from two unique experimental settings, specifically those with and without FLC administration, a notable disparity in the control of gene expression between these two situations was identified. In the context of the isw1 deletion strain exposed to FLC treatment, a set of 21 genes, including those belonging to the ABC/MFS family and efflux pumps, displayed significant changes in their gene expression patterns. In particular, a total of 9 genes exhibited downregulation, whilst 12 genes displayed upregulation. In contrast, in the absence of FLC supplementation, a total of 9 genes exhibited alterations in gene expression, with 3 genes showing downregulation and 6 genes showing upregulation. Therefore, the Isw1 protein plays a crucial role in the activation of certain genes, while simultaneously having a suppressive effect on other genes. Hence, the Isw1 undergoes a reconfiguration of its regulatory apparatus in response to drugs. Despite that the performance of ChIP-seq analysis was necessary in this study, it was observed that the treatment of fungal cells resulted in a notable decrease in the abundance of the Isw1 protein. This decrease can be attributed to the activation of Isw1 protein degradation. Consequently, there was an insufficient amount of Isw1 protein available for successful enrichment and subsequent ChIP-seq analysis (please see Figure 4a and 4c). However, the data collected collectively have demonstrated the idea that Isw1 serves as a crucial master regulator of drug resistance in C. neoformans. The text has undergone revisions in order to present our findings in a precise and thorough manner. Please see Figure 1c, 1g, 4a, 4b, Supplementary File 2, and line 145-153, 186-192, 256-260.

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[Editors’ note: what follows is the authors’ response to the second round of review.]

While the manuscript has been improved there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

General comments on revision: The revised manuscript is much improved overall. The authors have performed numerous additional experiments that strengthen the study. The majority of my initial comments have been addressed, and I thank the authors for carefully considering the comments and their responses. However, there are a few key points in the manuscript (largely associated with revisions or new data) that should be addressed to further strengthen the study and broaden its impact.

We express our sincere gratitude for your insightful feedback, particularly concerning the ISWI complex, which significantly contributes to the manuscript's improvement and facilitates readers' comprehension.

Prior reviewer comment: The authors demonstrate that Isw1 has a role in responding to antifungals in Cryptococcus. However, it is not clear if changes in Isw1 stability represent a general response to stress. This study would have benefited from experiments to test: (1) if levels of Isw1 change in response to other stressors (e.g., heat, osmotic, or oxidative stress) and (2) if loss of Isw1 impacts resistance to other stressors.

Author response: A series of experiments were conducted to illustrate and measure phenotypic traits associated with virulence. These traits encompassed capsule formation, melanin synthesis, cell proliferation under stressful conditions, and Isw1 expression levels in response to diverse environmental stimuli. Please see Figure 3a, 3b, 3c, Figure 3—figure supplement 1 and line 237-241.

Reviewer response: The authors provide new data to address the initial comment. The phenotypic data presented in Figure 3a, 3b, and 3c are convincing, although it should be noted that there appears to be a slight reduction in melanin production in Figure 3b. The data presented in Figure 3 —figure supplement 1 is less convincing. It is likely difficult to say "that Isw1 expression is not affected by external stress inducers" (line 237) considering data presented in Figure 1 —figure supplement 1 and Figure 4. Specifically, (1) ISW1 expression is not affected by antifungal treatment under the conditions tested and (2) changes in Isw1 levels associated with antifungal treatment are observed after inhibiting protein synthesis. It is thus plausible that changes in Isw1 levels under stress conditions could be missed in the assay presented in Figure 3 —figure supplement 1. The authors should change the text accordingly.

We highly appreciate your significant comment regarding the regulation of Isw1 protein levels in response to environmental conditions. In the previous revision, we performed protein quantification on Isw1-Flag under several stimuli and found that the total protein levels remained unchanged. Although we acknowledge that this experiment did not accurately represent the stability of the Isw1 protein under these specific conditions, the lack of any observed cell growth abnormalities in the spotting assays indicates that Isw1 is unlikely to play a significant role in controlling cellular responses to these stresses. As a result, we opted not to allocate additional resources towards conducting a more comprehensive investigation into the stability of Isw1 when protein synthesis is repressed. Additionally, we agree with the data about a slight decrease in melanin synthesis. The Results section has been modified to provide a more precise depiction of the findings. In addition, the Discussion section has been revised to point out the unique function of Isw1 in controlling fungal virulence factors and pathogenicity. It also emphasizes the significance of Isw1 in regulating drug resistance in both laboratory conditions and animal models. Please see lines 261-264, 267-269 and 496-507.

Prior reviewer comment: The authors find that deletion of ITC1, which is known to interact with Isw1 homologs in other systems, results in increased antifungal resistance. However, loss of Itc1 does not fully phenocopy loss of Isw1 (see Figure 1 —figure supplement 2 panel B). This is likely due to the activity of other Isw1 interacting proteins. The authors are encouraged to review recent literature on other organisms (particularly other fungi, such as Neurospora crassa) on other potential interacting proteins.

Author response: We acknowledge the significance of the conservation of Isw1 across many species. The regulatory pathway of Isw1 was initially discovered in the model organism Saccharomyces cerevisiae, which possesses two paralogs, namely Isw1 and Isw2, as a result of a whole genome duplication event(Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997). Due to the absence of a complete genome duplication event in C. neoformans, its genome solely contains a single copy of the ISW gene. Prior research conducted on Saccharomyces cerevisiae has provided evidence that the ISWI complex is composed of several subunits, namely Isw1, Ioc proteins, Itc1, Chd1, and Sua7 (Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013). The genome of C. neoformans was examined, and it was observed that the IOC gene family could not be identified. This suggests that the IOC gene family has likely undergone an evolutionary loss in C. neoformans, as indicated on the FungiDB website. The genome of C. neoformans harbors three distinct genes, namely Itc1, Chd1, and Sua7. In order to comprehensively investigate the cryptoccocal ISWI complex, we conducted a methodical Isw1-Flag protein immunoprecipitation procedure, which was subsequently followed by Mass-Spec analysis. In the present study, a total of 22 proteins that interacted with Isw1 were discovered. Among these proteins, 11 have been previously reported to be associated with the regulatory networks including Isw1. In the mass spectrometry results, Itc1 was found to be co-immunoprecipitated with Isw1. Although the Mass-Spec analysis did not reveal the presence of Chd1 and Sua7, our study demonstrated that Chd1 can be coimmunoprecipitated with Isw1 through co-IP and immunoblotting techniques. However, no interaction between Isw1 and Sua7 was established utilizing any of these methods. In order to gain a deeper understanding of the involvement of Chd1 and Itc1 in the regulation mechanism of Isw1 in multidrug resistance, we created strains with disrupted chd1Δ and itc1Δ genes. The only strain that exhibited a drug resistance phenotype similar to that of the isw1Δ strain was the itc1Δ strain. The data presented in this study indicate that there has been evolutionary divergence in the Isw1-protein complexes between the species C. neoformans and S. cerevisiae. The manuscript has undergone significant modifications. Please see Figure 2 and text line 208-232.

Reviewer response: The authors provide new data to data address the initial comment. This data greatly improves the manuscript. However, it also raises multiple concerns that must be addressed.

1. The authors should be mindful that (1) there is not a single "ISWI Complex" in S. cerevisiae or other eukaryotes, but rather ISWI homologs form multiple complexes (e.g., Isw1a, Isw1b, and Isw2 in S. cerevisiae); and (2) the IOC (Iswi One Complex) genes encoding Ioc2, Ioc3, and Ioc4 are not a "gene family" per se as the proteins are quite different, and it may be difficult to readily identify divergent homologs in C. neoformans. Additionally, the authors should clarify what is meant by "canonical ISWI complex" (line 231).

We deeply appreciate your perceptive feedback, especially about the ISWI complex, as it greatly enhances the text and aids readers in understanding. The amended text now includes a comprehensive background explanation of the ISWI complex in several eukaryotes, including the suggested fungus species Neurospora crassa. This addition allows for a more thorough understanding of the ISWI complex homologs in fungi. After considering the Ioc comments, we conducted a thorough sequence analysis, examining both sequence homology and gene synteny. With the assistance of a reputable fungal genome database (http://fungidb.org), we discovered that the C. neoformans genome does not possess the ioc2, ioc3, and ioc4 genes. Please consult below for the screenshot from the fungal database, which depicts the Ioc2 as an exemplar and reveals that only 9 orthologs were detected in fungi. Same analyses were also performed for Ioc3 and Ioc4 (screenshots not shown). Thirty-five orthologs were found for Ioc3, which corresponds to Gene YFR013W according to fungidb.org. Additionally, twelve orthologs were found for Ioc4, corresponding to Gene YMR044W according to fungidb.org. Nevertheless, the ortholog lists for these three proteins did not include any transcripts from C. neoformans. Thus, it is probable that C. neoformans had gene loss in the IOC genes during its evolutionary process, while S. cerevisiae and other fungal species potentially acquired additional copies of IOC genes. We concur with the reviewer's assertion that studying the intricate ISWI holds significant value in elucidating the evolutionary processes and governing cellular reactions. Nevertheless, we have conducted the first investigation of the ISWI complex in C. neoformans, and there remain several unanswered questions and intriguing phenomena that require more examination and analysis. The study establishes a connection between post-translational regulation and antifungal drug resistance in Cryptococcus neoformans, thereby uncovering a previously unknown aspect of the emergence of drug resistance. We contend that the absence of IOC genes in the genome of C. neoformans does not yield a significant conceptual breakthrough in establishing definitive proof of the connection between Isw1 PTM, its function, and drug resistance. While we concur with the reviewer's opinion that studying the components and mechanisms of Isw1 regulation offers valuable insights into how C. neoformans responds to anti-fungal treatment and is relevant for clinical therapy, our future analysis aims to delve deeper into this intricate regulatory process of Isw1. We have made significant revisions to the manuscript, namely altering the description of what is considered "canonical". Please see lines 256-259.

Author response image 4

2. It is unclear why the authors chose to focus on Chd1 and Sua7 as (1) neither Chd1 nor Sua7 appear in the AP-MS data and (2) there is very little evidence that Chd1 or Sua7 directly interact with Isw1 or Isw2 in S. cerevisiae (to the best of my knowledge, Michaelis et al., 2023 Nature provides the only biochemical evidence for interaction between Isw1 and Chd1 in S. cerevisiae). There is evidence that both Chd1 and Sua7 genetically interact with Isw1 or Isw2 in S. cerevisiae, which is what is described in the references cited by the authors (for example, Smolle et al., 2012). Along these lines, the authors should review the text and references and revise both accordingly. It should be noted that the evidence for an association between Isw1 and Chd1 in C. neoformans is quite interesting (although it may not be a direct physical interaction).

We really appreciate your feedback and sincerely apologize for erroneously identifying Chd1 and Sua7 as the interacting proteins with Isw1 from the literatures. After taking into account these crucial remarks, we have thoroughly examined the reference. There is no empirical data indicating that Chd1 and Sua7 have a protein-protein interaction with Isw1. In the study conducted by Smolle et al. in 2012, it was found that Chd1 and Isw1 had similar transcriptional controls on specific genes in the Set2 pathway. Consequently, this presented a compelling rationale and facet to investigate whether the simultaneous regulation of drug resistance occurs by C. neoformans Chd1 and Isw1. Our findings suggest that the control of drug resistance in C. neoformans is mediated by the Isw1-Itc1 complex pathway, rather than the Isw1-Chd1 pathway. Based on the Sua7 assays, there is currently no definitive evidence regarding the interaction. However, it has been observed that Sua7 mutants can influence the DNA binding targets of Isw2. It is therefore our primary intention to conduct experiments on these proteins. We concur with the reviewer's comments that these interactions reveal intriguing facets of Isw1's control. We intend to thoroughly examine them in the ongoing laboratory analysis to gather more detailed data for future analysis. The text has been altered, please see lines 135-139, 241-250, 471-474, and 479-482.

3. As mentioned in the initial comment, the authors are encouraged to review recent literature on ISWI homologs in organisms other than S. cerevisiae (e.g., Neurospora crassa – Kamei et al., 2021 PNAS and Wiles et al., 2022 eLife) to strengthen this aspect of the manuscript. For example, there are at least two additional proteins in the AP-MS data that are well-characterized to interact with ISWI homologs. Along these lines, the authors should provide protein names in addition to locus IDs in Figure 2a (as was done for Itc1) to help readers and broaden interest.

We concur with the comment. We have enhanced the introduction section by include additional details regarding ISWI complexes derived from various organisms. The modification of Figure 2a now includes gene names, so enhancing the provision of specific information to readers, please see lines 115-139, 241-242 and 1264.

Prior reviewer comment: The authors' use of "increase", "induce", or similar wording is confusing in some instances. For example, in Lines 197-198 the authors suggest that deacetylase inhibitors "increase acetylation levels" (see also Lines 235, 283, and 284 for additional examples). The authors should consider rewording these examples and reviewing the manuscript for similar examples to clarify their results.

Author response: The manuscript has been appropriately updated in accordance with the given instructions.

Reviewer response: Many such instances were not corrected (for example, see lines 281 and 322-323). Similarly, (1) in lines 127-128, the authors state "Cryptococcal Isw1 plays an indispensable role in modulating drug-resistance genes", but the role of Isw1 is not indispensable since the isw1 mutant strain has increased resistance to antifungals; and (2) in line 247, the authors state "that the isw1 mutant strain exhibits improved fungal burdens", but the isw1 mutant strain showed increased fungal burdens not improved fungal burdens.

We appreciate your thoughtful evaluations and comments. We agree that the aforementioned terms are inadequate in characterizing the data. They have been adjusted accordingly. Please see lines 309, 350-351, 153 and 275-276.

https://doi.org/10.7554/eLife.85728.sa2

Article and author information

Author details

  1. Yang Meng

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
  2. Yue Ni

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Data curation, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  3. Zhuoran Li

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Tianhang Jiang

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Data curation, Methodology
    Competing interests
    No competing interests declared
  5. Tianshu Sun

    Department of Scientific Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
    Contribution
    Conceptualization, Project administration
    Competing interests
    No competing interests declared
  6. Yanjian Li

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Conceptualization, Methodology
    Competing interests
    No competing interests declared
  7. Xindi Gao

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Software, Visualization, Methodology
    Competing interests
    No competing interests declared
  8. Hailong Li

    NHC Key Laboratory of AIDS Immunology, The First Affiliated Hospital of China Medical University, Shenyang, China
    Contribution
    Software, Funding acquisition, Methodology
    Competing interests
    No competing interests declared
  9. Chenhao Suo

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  10. Chao Li

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  11. Sheng Yang

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  12. Tian Lan

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Software
    Competing interests
    No competing interests declared
  13. Guojian Liao

    College of Pharmaceutical Sciences, Southwest University, Chongqing, China
    Contribution
    Resources, Writing - review and editing
    Competing interests
    No competing interests declared
  14. Tongbao Liu

    Medical Research Institute, Southwest University, Chongqing, China
    Contribution
    Resources, Writing - review and editing
    Competing interests
    No competing interests declared
  15. Ping Wang

    Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center New Orleans, New Orleans, United States
    Contribution
    Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
  16. Chen Ding

    College of Life and Health Sciences, Northeastern University, Shenyang, China
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    dingchen@mail.neu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9195-2255

Funding

National Key Research and Development Program of China (2022YFC2303000)

  • Chen Ding

National Natural Science Foundation of China (31870140)

  • Chen Ding

Liaoning Revitalization Talents Program (XLYC1807001)

  • Chen Ding

National Institutes of Health (AI156254)

  • Ping Wang

National Institutes of Health (AI168867)

  • Ping Wang

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Profs. Yongqiang Fan and Ren Sheng for their critical review of the manuscript. This work was supported by the National Key Research and Development Program of China (2022YFC2303000). Funds for this program were also provided by the National Natural Science Foundation of China (31870140 to CD) and Liaoning Revitalization Talents Program (XLYC1807001 to CD). Research in PW lab was supported by the National Institutes of Health (US) awards AI156254 and AI168867.

Ethics

All animal experiments were reviewed and ethically approved by the Research Ethics Committees of the National Clinical Research Center for Laboratory Medicine of the First Affiliated Hospital of China Medical University (KT2022284) and were carried out in accordance with the regulations in the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China. Infections with C. neoformans were performed via the intranasal route. Four- to six-week-old female Balb/c mice were purchased from Changsheng Biotech (Liaoning, China) and used for survival and fungal burden analyses.

Senior and Reviewing Editor

  1. Detlef Weigel, Max Planck Institute for Biology Tübingen, Germany

Version history

  1. Received: December 21, 2022
  2. Preprint posted: December 30, 2022 (view preprint)
  3. Accepted: January 21, 2024
  4. Accepted Manuscript published: January 22, 2024 (version 1)
  5. Version of Record published: February 1, 2024 (version 2)

Copyright

© 2024, Meng et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Yang Meng
  2. Yue Ni
  3. Zhuoran Li
  4. Tianhang Jiang
  5. Tianshu Sun
  6. Yanjian Li
  7. Xindi Gao
  8. Hailong Li
  9. Chenhao Suo
  10. Chao Li
  11. Sheng Yang
  12. Tian Lan
  13. Guojian Liao
  14. Tongbao Liu
  15. Ping Wang
  16. Chen Ding
(2024)
Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans
eLife 13:e85728.
https://doi.org/10.7554/eLife.85728

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https://doi.org/10.7554/eLife.85728

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