The acetylase activity of Cdu1 regulates bacterial exit from infected cells by protecting Chlamydia effectors from degradation

eLife assessment

This important study combines state-of-the art proteomics and genetic manipulation of Chlamydia trachomatis to study the function of a chlamydial effector, Cdu1, with deubiquitination and acetylation activities. Solid evidence is provided to show that Cdu1 is able to protect itself and three other chlamydial effectors, which are involved in the control of chlamydial egress from host cells, from ubiquitin-mediated degradation, and that this depends on the acetylation activity of Cdu1, but not on its deubiquitination activity. This work will be of interest to microbiologists and cell biologists studying host cell-pathogen interactions.

https://doi.org/10.7554/eLife.87386.3.sa0

Significance of the findings:

Important: Findings that have theoretical or practical implications beyond a single subfield

Landmark
Fundamental
Important
Valuable
Useful

Strength of evidence:

Solid: Methods, data and analyses broadly support the claims with only minor weaknesses

Exceptional
Compelling
Convincing
Solid
Incomplete
Inadequate

During the peer-review process the editor and reviewers write an eLife Assessment that summarises the significance of the findings reported in the article (on a scale ranging from landmark to useful) and the strength of the evidence (on a scale ranging from exceptional to inadequate). Learn more about eLife Assessments

Abstract
Introduction
Results
Discussion
Materials and methods
Appendix 1
Data availability
References
Article and author information
Metrics

Abstract

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen Chlamydia trachomatis secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins. Here, we determined that Cdu1’s acetylase but not its DUB activity is important to protect Cdu1 from ubiquitin-mediated degradation. We further identified three C. trachomatis proteins on the pathogen-containing vacuole (InaC, IpaM, and CTL0480) that required Cdu1‘s acetylase activity for protection from degradation and determined that Cdu1 and these Cdu1-protected proteins are required for optimal egress of Chlamydia from host cells. These findings highlight a non-canonical mechanism of pathogen-mediated protection of virulence factors from degradation after their delivery into host cells and the coordinated regulation of secreted effector proteins.

Introduction

Ubiquitination is a conserved and ubiquitous post-translational modification (PTM) of proteins involving the conjugation of the carboxy-terminal glycine residue of ubiquitin (Ub) to lysine residues of target proteins. Poly-ubiquitination of substrates involves further conjugation of a Ub internal lysine residue or amino-terminal methionine (M1) with a second Ub molecule. Seven internal lysines in Ub (K6, K11, K27, K29, K33, K48, K63) and M1 are utilized by Ub conjugating enzymes to form homogeneous, branched, or mixed poly-ubiquitin (polyUb) chains (Komander and Rape, 2012). PolyUb chains with different linkage types exhibit distinct structures and functions. For example, K48- and K11-linked polyUb chains exhibit a compact conformation and are substrates for 26 S proteasome-mediated degradation (Varadan et al., 2002; Tenno et al., 2004; Eddins et al., 2007; Bremm et al., 2010; Saeki, 2017). In contrast, K63-linked polyUb conjugates adopt more open conformations that enable the recruitment of multiprotein complexes that regulate the function of the target protein by proteolytic independent events (Komander et al., 2009b; Weeks et al., 2009; Datta et al., 2009; Komander and Rape, 2012). Mixed and branched polyUb chains are also emerging as important regulators of physiological functions (Swatek and Komander, 2016; Ohtake and Tsuchiya, 2017).

Protein ubiquitination regulates numerous eukaryotic cell processes including protein degradation, signal transduction, cell cycle regulation, selective autophagy, the DNA damage response, and programmed cell death. Ub also plays key roles in modulating host innate immune responses to bacterial infection (Li et al., 2016), bacterial proteins, pathogen-containing vacuoles, and bacteria themselves by targeting them for Ub-mediated degradation by proteasomal or autophagic machineries (Li et al., 2016). Because the Ub system is critical for pathogen containment, many pathogens have evolved mechanisms to counteract the impact of this PTM (Vozandychova et al., 2021). For instance, bacterial deubiquitinases (DUBs) can remove Ub from ubiquitinated substrates thereby dampening inflammatory and cell-autonomous defense mechanisms (Kubori et al., 2019). Many DUBs are cysteine proteases with a catalytic Cys, a nearby His and an Asn/Asp (Komander et al., 2009a). DUBs are typically dedicated to the removal of Ub moieties and are unable to hydrolyze other Ub-like (Ubl) modifications such as SUMO or NEDD8. However, the CE clan of Ubl proteases (ULPs) can catalyze the removal of both SUMO and NEDD8 (Ronau et al., 2016). Bacterial pathogens also encode CE clan enzymes that function as DUBs, ULPs or both. For instance, Salmonella Typhimurium SseL, Escherichia coli ElaD, and Shigella flexneri ShiCE function as Ub specific proteases (Rytkönen et al., 2007; Catic et al., 2007; Pruneda et al., 2016) while RickCE from Rickettsia belli functions as a protease directed towards both Ub and NEDD8 as does SidE from Legionella pneumophila which displays mixed activities towards Ub, NEDD8, and ISG15 (Sheedlo et al., 2015; Pruneda et al., 2016). Similarly, XopD from Xanthamonas campestris and LotB from L. pneumophila are isopeptidases exhibiting cross reactivity towards both Ub and SUMO (Pruneda et al., 2016; Schubert et al., 2020). Some CE clan bacterial effectors display acetyltransferase activity. L. pneumophila LegCE, S. Typhimurium AvrA, and YopJ from Yersinia pestis function exclusively as acetyltransferases (Mittal et al., 2006; Mukherjee et al., 2006; Jones et al., 2008; Pruneda et al., 2016). In contrast, the Chlamydia trachomatis (Ct) effector Cdu1/ChlaDUB1 is a CE clan protein that exhibits both acetyltransferase and deubiquitinating activities (Misaghi et al., 2006; Pruneda et al., 2016; Fischer et al., 2017; Pruneda et al., 2018).

Ct is an obligate intracellular bacterial pathogen responsible for human diseases of significant clinical and public health importance (Haggerty et al., 2010). Ct has a biphasic developmental cycle in which the Ct infectious propagule or elementary body (EB) invades the target host cell. Upon internalization the EB transitions to the reticulate body (RB). RBs replicate by binary fission within a pathogenic vacuole (‘inclusion’) and asynchronously differentiate back to EBs. In cell culture, starting at around 48 hours-post infection (hpi) Ct will egress after lysis of the host cell or by a process termed extrusion, wherein the intact inclusion exits from the infected cell (Moulder, 1991; Abdelrahman and Belland, 2005; Hybiske and Stephens, 2007; Lee et al., 2018). The effector Cdu1 was originally identified as a deneddylating and deubiquitinating enzyme and subsequently shown to exhibit in vitro isopeptidase activity towards both Lys48 and Lys63 linked di-Ub substrates (Misaghi et al., 2006; Claessen et al., 2013; Pruneda et al., 2016; Fischer et al., 2017). Cdu1 is unique among CE clan enzymes in that it also functions as a bona fide lysine acetylase with both is acetylase and DUB activities catalyzed by the same catalytic active site (Pruneda et al., 2018). Intriguingly, Cdu1 autoacetylation is directed towards lysines unlike other CE clan acetylases that predominantly target serine and threonine residues (Pruneda et al., 2018). In transfected cells, Cdu1 protects the NFκB cytoplasmic retention factor IκBα from ubiquitination and proteasomal degradation (Le Negrate et al., 2008). In infected cells, Cdu1 localizes to the inclusion membrane where it functions to stabilize the anti-apoptotic protein Mcl-1 and to promote the repositioning of Golgi ministacks around the Ct inclusion (Fischer et al., 2017; Wang et al., 2018; Pruneda et al., 2018; Kunz et al., 2019; Auer et al., 2020). However, the mechanism by which Cdu1 promotes redeployment of Golgi ministacks and any additional roles that Cdu1 may play during Ct infection of epithelial cells remains unknown.

In this study, we show that Cdu1 protects itself and three secreted Ct effectors, InaC, IpaM, and CTL0480 from targeted ubiquitination and proteasomal degradation. InaC, IpaM, and CTL0480 are members of a larger family of bacterial proteins embedded within the inclusion membrane (Inc proteins; Bannantine et al., 2000; Rockey et al., 2002; Chen et al., 2006; Li et al., 2008; Alzhanov et al., 2009; Dehoux et al., 2011; Lutter et al., 2012; Lutter et al., 2013; Kokes et al., 2015; Weber et al., 2015). We show that Cdu1-mediated protection from degradation is independent from its DUB activity but relies upon its Lys acetylase activity. We show that Cdu1 protects InaC to promote repositioning of Golgi ministacks and formation of actin scaffolds around the Ct inclusion, and CTL0480 to promote recruitment of myosin phosphatase target subunit 1 (MYPT1) to the inclusion. In addition, we determined that Cdu1 and Cdu1-protected Incs are required for optimal extrusion of inclusions from host cells at the late stages of infection.

Results

The C. trachomatis inclusion membrane proteins InaC, IpaM, and CTL0480 are differentially ubiquitinated in the absence of Cdu1

Cdu1 is required for Golgi repositioning around the Ct inclusion (Pruneda et al., 2018; Auer et al., 2020). To understand how Cdu1 promotes Golgi redistribution, we first generated a cdu1 null strain in a Ct L2 background by TargeTron mediated insertional mutagenesis (pDFTT3-aadA) (Lowden et al., 2015; Figure 1—figure supplement 1). Loss of Cdu1 expression in the resulting L2 cdu1::GII aadA (cdu1::GII) strain was verified by western blot analysis and by indirect immunofluorescence with antibodies raised against Cdu1 (Figure 1—figure supplement 2A and B). Because cdu2 resides directly downstream of the cdu1 locus and encodes a second Ct DUB (Cdu2/ChlaDUB2) (Misaghi et al., 2006), we first determined whether the disruption of cdu1 impacted the expression of cdu2. We detected cdu1 and cdu2 transcripts in HeLa cells infected with Ct L2 but not for the juncture between cdu1 and cdu2 (Figure 1—figure supplement 2C). In cells infected with Ct cdu1::GII we only detected cdu2 transcripts (Figure 1—figure supplement 2C) confirming that cdu1 and cdu2 are not co-expressed as part of an operon in accordance with previous observations (Albrecht et al., 2010).

In transfected HeLa cells, Cdu1’s DUB activity has been linked to fragmentation of the Golgi apparatus (Pruneda et al., 2018). We therefore hypothesized that Cdu1s’ DUB activity in infected cells promoted Golgi redistribution around inclusions and that we could identify potential targets by comparing the protein ubiquitination profile of cells infected with WT or cdu1::GII strains by quantitative mass spectrometry (MS). HeLa cell were mock infected or infected with either WT L2 or cdu1::GII strains. At 24 hpi, poly-ubiquitinated proteins were enriched from lysed cells using Tandem Ubiquitin Binding Entities (TUBEs; LifeSensors). TUBEs consist of concatenated Ub binding associated domains (UBAs) that bind to polyUb-modified proteins with nanomolar affinities. Poly-ubiquitinated proteins of both human and Ct origin were enriched and identified by quantitative LC-MS/MS analysis.

Over 2000 non-ubiquitinated proteins co-precipitated with TUBE1-bound proteins across all three conditions (mock, L2, and cdu1::GII infected HeLa cells) and three biological replicates (Supplementary file 1). Among these, 47 human proteins were significantly enriched in mock infected HeLa cells and 50 human proteins were significantly enriched during Ct infection (L2 and cdu1::GII) (Supplementary file 3, Figure 1—figure supplement 3). Pathway enrichment analysis revealed that proteins involved in RNA metabolism were overrepresented among co-precipitating proteins from mock infected cells (Supplementary file 4, Figure 1—figure supplement 4) while no biological pathways or processes were overrepresented in proteins enriched from infected cells (Figure 1—figure supplement 4). We also identified eight TUBE1 co-precipitating Ct proteins in HeLa cells infected with L2 and cdu1::GII (Supplementary file 5, Figure 1—figure supplement 3).

TUBE1 affinity capture lead to the identification of 43 ubiquitinated proteins (35 human proteins and 8 Ct proteins across all 3 conditions and replicates) based on the presence of peptides containing a di-glycine remnant motif (Peng et al., 2003; Supplementary files 6-8). The lack of widespread poly-ubiquitination of either human or Ct proteins in response to Ct infection (Figure 1) was surprising given that wholesale changes in protein ubiquitination has been reported during infection of HeLa cells by intracellular pathogens like S. Typhimurium (Fiskin et al., 2016). It is also possible that had we conducted our analysis at different time points post-infection (hpi), we might have identified additional Cdu1 targets, such as Mcl1 and IκBα (Le Negrate et al., 2008; Fischer et al., 2017) which were not identified in our analysis. However, given that we observed Cdu1 at the inclusion membrane as early as 1 hpi (data not shown), we opted to focus on an earlier stage of the infection cycle. Only two human ubiquitinated proteins (ZC3H7A and DDIT4) were found to be significantly enriched in response to WT L2 infection at 24 hpi (Figure 1A and C, Supplementary file 8) while only one human protein (MGC3121) was preferentially ubiquitinated in HeLa cells infected with the cdu1::GII mutant strain (Figure 1B and C, Supplementary file 8). In contrast three Ct proteins, InaC (K104, K107, and K149), IpaM (K29), and CTL0480 (K115) were ubiquitinated at Lys residues in the absence of Cdu1 (Figure 1B, C and D, Supplementary file 8).

Figure 1 with 4 supplements see all

Download asset Open asset

The C. *trachomatis* inclusion membrane proteins InaC, IpaM, and CTL0480 are ubiquitinated in the absence of Cdu1.

(**A–C**) Volcano plots (pairwise comparisons) of the relative abundance of human and Ct ubiquitinated peptides.(A) Mock infected HeLa cells versus HeLa cells infected with WT Ct (L2 434 Bu pBOMB) (24 hpi). (B) Mock infected HeLa cells versus HeLa cells infected with a *cdu1* null strain (*cdu1*::GII pBOMB) (24 hpi) (C) HeLa cells infected with WT Ct (24 hpi) versus HeLa cells infected with a *cdu1* null strain (24 hpi). Significance values were interpolated from 3 independent biological replicates. Ubiquitinated proteins were enriched with magnetic TUBE1 beads (binds to polyubiquitinated proteins) and peptides identified by quantitative LC MS/MS analysis. Three Ct inclusion membrane proteins, InaC, IpaM, and CTL0480 were differentially ubiquitinated in the absence of Cdu1. (D) InaC was ubiquitinated at K104, K107, and K149, IpaM at K290, and CTL0480 at K115 in the absence of Cdu1. TMD: Transmembrane domain. Numbering corresponds to amino acids in the protein sequence of each respective inclusion membrane protein.

Figure 1—source data 1 Excel file containing MaxQuant intensity values used to generate volcano plots in Figure 1A–C.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig1-data1-v1.zip
Download elife-87386-fig1-data1-v1.zip

InaC, IpaM, and CTL0480 are Ct effector proteins that localize to the inclusion membrane (Chen et al., 2006; Alzhanov et al., 2009; Lutter et al., 2013; Kokes et al., 2015). These Type 3 secretion substrates belong to a family of over 36 inclusion membrane proteins (Incs) that contain a signature bi-lobal hydrophobic transmembrane domain (Bannantine et al., 2000; Rockey et al., 2002; Li et al., 2008; Dehoux et al., 2011; Lutter et al., 2012). Incs provide many functions important for Ct intracellular replication ranging from providing structural integrity to the inclusion membrane, to regulating membrane trafficking, to mediating interactions with host organelles and cytoskeletal structures (reviewed in Bugalhão and Mota, 2019). InaC facilitates the activation of the small GTPase RhoA, a crucial step for the assembly of actin scaffolds around the inclusion (Haines et al., 2021; Kumar and Valdivia, 2008). Additionally, InaC plays a pivotal role in the activation of Arf GTPases. This activation subsequently induces PTMs of microtubules in close proximity to the inclusion membrane, which are essential for Ct to initiate the repositioning of Golgi ministacks around the inclusion (Wesolowski et al., 2017). IpaM exhibits localization to discrete patches in the inclusion termed microdomains (Alzhanov et al., 2009; Dumoux et al., 2015). Upon ectopic expression, IpaM induces alterations in microtubule organization (Dumoux et al., 2015). CTL0480, facilitates the recruitment of MYPT1 (myosin phosphatase target subunit 1) to the inclusion membrane. Recrutiment of MYPT1 is required for the efficient exit of Ct from host cells (Lutter et al., 2013; Shaw et al., 2018). Because Cdu1 also localizes to the inclusion membrane (Fischer et al., 2017; Wang et al., 2018; Pruneda et al., 2018; Kunz et al., 2019) we postulated that Cdu1 directly protects InaC, IpaM, and CTL0480 from ubiquitination.

Cdu1 associates with InaC, IpaM, and CTL0480

We first determined if Cdu1 co-localized with InaC, IpaM, and CTL0480 at the inclusion membrane. HeLa cells were infected for 24 hr with WT L2 or L2 expressing CTL0480-Flag from its endogenous promoter and immunostained with antibodies against Cdu1, InaC, IpaM, or the Flag epitope. Both InaC and CTL0480-Flag localized throughout the inclusion membrane while IpaM was restricted to discrete microdomains as previously reported (Alzhanov et al., 2009; Dumoux et al., 2015; Figure 2A). Cdu1 co-localized with InaC and CTL0480-Flag and with IpaM at microdomains (Figure 2A and B). All four antibodies specifically recognized their corresponding antigens since immunostaining for Cdu1, InaC, IpaM or the Flag epitope was not observed in Ct strains lacking Cdu1 (cdu1::GII), InaC (M407, Kokes et al., 2015) IpaM (ipaM::GII, Meier et al., 2023), or a strain that does not express CTL0480-Flag (Figure 2A).

Figure 2

Download asset Open asset

Cdu1 associates with InaC, IpaM, and CTL0480.

(A) Co-localization of Cdu1(magenta) with endogenous InaC (green), IpaM (green), and ectopically expressed CTL0480-Flag (green) at the Ct (L2) inclusion membrane of HeLa cells infected for 24 hr. HeLa cells infected with an *inaC* null strain (M407), an *ipaM* null strain (*ipaM*::GII), and WT Ct (L2 434 Bu pBOMB) were used as controls for antibody specificity. DNA stained with Hoechst is shown in blue. Scale bar: 10 μm. Images are representative of multiple images captured across three independent replicates. (B) Line scan profiles of fluorescent signal intensities displayed in (A) showing co-localization of fluorescence intensities for endogenous Cdu1 with endogenous InaC and IpaM, and with CTL0480-Flag along the L2 inclusion membrane. (C) Schematic of Cdu1-GFP(C) (L2) fusion (Cdu1-GFP) and Cdu1-GFP variants used in co-transfections of HEK 293 cells. GFP: Green fluorescent protein. TMD: Transmembrane domain. PRD: Proline rich domain. CD: Catalytic domain. FL: Full length. TMD-: Cdu1-GFP variant lacking TMD domain. CD-: Cdu1-GFP variant lacking CD domain. (**D–G**) Western blot analysis of GFP immunoprecipitates from HEK 293 cells co-transfected with mammalian plasmids expressing: Cdu1-GFP variants and (D) truncated 3XFlag(N)-InaC (D/UW-3/CX CT813, amino acids 96–264), (E) V5(N)-IpaM (L2, full length), (F) V5(N)-CTL0480 (L2, full length), and (G) V5(N)-CpoS (L2, full length). Vec1: Empty pOPINN-GFP vector. Vec2: Empty pDEST53 vector. Vec3: Empty pcDNA3.1/nV5-DEST vector. Western blot images are representative from two independent experiments.

Figure 2—source data 1 Excel files with pixel intensity values for generating line scan profiles in Figure 2B.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data1-v1.zip
Download elife-87386-fig2-data1-v1.zip
Figure 2—source data 2 Original western blot scans used to generate Figure 2D (anti-GFP [input], anti-Flag [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-Flag [IP:GFP]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data2-v1.zip
Download elife-87386-fig2-data2-v1.zip
Figure 2—source data 3 Original western blot scans with labeled samples and highlighted bands for Figure 2D.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data3-v1.zip
Download elife-87386-fig2-data3-v1.zip
Figure 2—source data 4 Original western blot scans used to generate Figure 2E (anti-GFP [input], anti-V5 [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-V5 [IP:GFP]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data4-v1.zip
Download elife-87386-fig2-data4-v1.zip
Figure 2—source data 5 Original western blot scans with labeled samples and highlighted bands for Figure 2E.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data5-v1.zip
Download elife-87386-fig2-data5-v1.zip
Figure 2—source data 6 Original western blot scans used to generate Figure 2F (anti-GFP [input], anti-V5 [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-V5 [IP:GFP]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data6-v1.zip
Download elife-87386-fig2-data6-v1.zip
Figure 2—source data 7 Original western blot scans with labeled samples and highlighted bands for Figure 2F.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data7-v1.zip
Download elife-87386-fig2-data7-v1.zip
Figure 2—source data 8 Original western blot scans used to generate Figure 2G (anti-GFP [input], anti-V5 [input], anti-α-tubulin [input], anti-GFP [IP:GFP], and anti-V5 [IP:GFP]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data8-v1.zip
Download elife-87386-fig2-data8-v1.zip
Figure 2—source data 9 Original western blot scans with labeled samples and highlighted bands for Figure 2G.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig2-data9-v1.zip
Download elife-87386-fig2-data9-v1.zip

We next determined if Cdu1 can interact with InaC, IpaM, and CTL0480 by co-transfecting HEK 293 cells with vectors expressing either full length Cdu1-GFP or truncated versions of Cdu1-GFP lacking transmembrane or catalytic domains (Figure 2C), and vectors expressing Flag-InaC, V5-IpaM, and V5-CTL0480. Transfected cells were lysed and Cdu1-GFP was immunoprecipitated with antibodies against GFP. Western blot analysis of the immunoprecipitates showed that Flag-InaC, V5-IpaM, and V5-CTL0480 co-precipitated with Cdu1-GFP (Figure 2D–F). Moreover, the transmembrane domain of Cdu1 was necessary for Cdu1-GFP to interact with all three Incs (Figure 2D–F). The interaction between Cdu1-GFP and the three tagged Incs was specific since we did not detect interactions between Cdu1-GFP and a V5-tagged version of the inclusion membrane protein CpoS (Sixt et al., 2017; Figure 2G). We expected these interactions to be transient at the inclusion membrane as the engagement of Cdu1 with its target(s) should mimic that of most enzymes with their substrates. Therefore, while we could capture these complexes in co-immunoprecipitations in the context of overexpression, this assay was not sensitive enough to reliably document the formation of complexes among low abundance endogenous Ct proteins. Nevertheless, our findings from transfection experiments lead us to conclude that Cdu1 can selectively interact with all three Incs even in the absence of infection, and these interactions are facilitated by the transmembrane domain of Cdu1.

Cdu1 protects InaC, IpaM, and CTL0480 proteins from degradation during infection

We next assessed if Cdu1 was required to stabilize endogenous InaC, IpaM, and CTL0480 in infected cells. HeLa cells were infected with either WT L2 or cdu1::GII strains and at various time points in the Ct infectious cycle crude cell lysates were analyzed by western blot to assess the relative abundance of Inc proteins. At 36 and 48 hpi, the levels of InaC protein were found to be undetectable in cells infected with cdu1::GII in comparison to cells infected with the WT L2 strain (Figure 3A and B). Conversely, IpaM protein levels exhibited a decrease at 36 and 48 hpi in cells infected with cdu1::GII (Figure 3C and D). We were not able to detect CTL0480 levels by western blot but were successful in following CTL0480 expression by indirect immunofluorescence (Figure 3E). The relative abundance of CTL0480 at inclusion membranes was not affected in cdu1::GII inclusions at 24 hpi. However, at 36 hpi, a subpopulation of cells lost CTL0480 immunoreactivity and by 48 hpi, inclusion membranes of the cdu1::GII strain were devoid of CTL0480 while CTL0480 was prominently detected at the inclusion membranes of WT L2 (Figure 3E and Figure 3—figure supplement 1). As controls for the specificity of antibodies used for western blots and for indirect immunofluorescence, we included cells infected with Ct lacking ipaM (ipaM::GII, Meier et al., 2023), with an inaC nonsense mutant (M407, Kokes et al., 2015), and with Ct lacking ctl0480 (ctl0480::GII, Shaw et al., 2018). Overall, our results indicate that steady state protein levels of InaC and IpaM and CTL0480 localization at the inclusion membrane, especially at late stages of infection, are dependent on Cdu1, and that Cdu1 acts at different stages in the infection cycle.

Figure 3 with 1 supplement see all

Download asset Open asset

Cdu1 stabilizes InaC, IpaM, and CTL0480.

(A) Western blot analysis of endogenous InaC in HeLa cells infected for 24, 36, and 48 hr with Wt Ct (L2 pBOMB), *cdu1* null (*cdu1*::GII pBOMB), and *inaC* null (M407) strains. Ct Slc1 and human alpha tubulin were used to determine Ct burdens and equal loading of protein extracts respectively. Western blot images are representative of 2 independent experiments. (B) Quantification of InaC abundance (InaC western blot signal from (A)) normalized to Slc1 western blot signal (from panel A) in HeLa cells infectedwith a *cdu1* null strain, relative to normalized InaC abundance in HeLa cells infected with Wt Ct. Error bars depict standard deviation. (C) Western blot analysis of endogenous IpaM in HeLa cells infected for 24, 36, and 48 hr with Wt Ct, *cdu1* null, and *ipam* null (*ipam*::GII) strains. Western blot images are representative of two independent experiments. (D) Quantification of normalized IpaM abundance from (C) in HeLa cells infectedwith a *cdu1* null strain, relative to normalized IpaM abundance in HeLa cells infected with Wt Ct. Error bars depict standard deviation. (E) Localization of CTL0480 during Ct infection of HeLa cells at 24, 36, and 48 hpi. CTL0480 signal (green) co-localizes with the inclusion membrane protein IncA (magenta) at the Ct inclusion membrane. Arrowheads highlight *cdu1* null inclusions lacking CTL0480 at 36 hpi. DNA stained with Hoechst is shown in blue. Scale bar: 10 μm. Quantification of CTL0480 localization at inclusion membranes can be found in Figure 3—figure supplement 1.

Figure 3—source data 1 Original western blot scans used to generate Figure 3A (anti-InaC, anti-Slc1, and anti-α-tubulin).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data1-v1.zip
Download elife-87386-fig3-data1-v1.zip
Figure 3—source data 2 Original western blot scans with labeled samples and highlighted bands for Figure 3A.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data2-v1.zip
Download elife-87386-fig3-data2-v1.zip
Figure 3—source data 3 Excel file containing densitometry data for Figure 3B.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data3-v1.zip
Download elife-87386-fig3-data3-v1.zip
Figure 3—source data 4 Original western blot scans used to generate Figure 3C (anti-IpaM, anti-Slc1, and anti-α-tubulin).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data4-v1.zip
Download elife-87386-fig3-data4-v1.zip
Figure 3—source data 5 Original western blot scans with labeled samples and highlighted bands for Figure 3C.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data5-v1.zip
Download elife-87386-fig3-data5-v1.zip
Figure 3—source data 6 Excel file containing densitometry data for Figure 3D.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig3-data6-v1.zip
Download elife-87386-fig3-data6-v1.zip

The acetylase activity of Cdu1 is required for Cdu1 to protect itself, InaC, and IpaM from polyubiquitination and proteasomal degradation

The crystal structures of Cdu1 bound to Ub or Coenzyme A indicated that the adenosine and phosphate groups of Coenzyme A make contact with a helix in variable region 3 (VR-3) of Cdu1 while the Ile36-patch of Ub binds to the opposite face of the same helix (Pruneda et al., 2018). Although Cdu1 catalyzes both of its DUB and acetylase (Act) activities with the same active site (Pruneda et al., 2018) the two activities of Cdu1 can be uncoupled by the amino acid substitution K268E in VR-3 which disrupts Coenzyme A binding required for Act activity and by the amino acid substitution I225A in the Ub-binding region of VR-3 required for DUB activity (Pruneda et al., 2018). These substitutions allowed us to test which of Cdu1’s enzymatic functions are required for the observed effects on protein stability. We generated Ct shuttle plasmids, expressing WT Cdu1, a catalytically inactive variant of Cdu1 lacking both DUB and Act activities (Cdu1^C345A; Pruneda et al., 2018), a Cdu1 DUB-deficient variant (Cdu1^I225A), and a Cdu1 Act-deficient variant (Cdu1^K268E). All Cdu1 constructs were expressed from the cdu1 endogenous promoter as 3 X Flag epitope-tagged proteins.

Plasmids expressing each Cdu1 variant were transformed into the cdu1::GII mutant and the resulting strains used to infect HeLa cells for 36 and 48 hr. The levels of endogenous InaC and IpaM in cell extracts of infected cells were monitored by western blot analysis. At 36 hpi, InaC protein levels drastically decreased in cells infected with cdu1 null strains transformed with empty vector or expressing the catalytic inactive variant of Cdu1 (Cdu1^C345A-Flag; Figure 4A). Likewise, IpaM protein levels diminished at 48 hpi during infection with the same strains (Figure 4B). Both InaC and IpaM protein levels were restored to wild type levels in cdu1 null strains complemented with wild type Cdu1-Flag (Figure 4A and B). Unexpectedly, cells infected with a cdu1 null strain ectopically expressing the DUB-deficient Cdu1 variant (Cdu1^I225A-Flag) displayed wild type levels of InaC and IpaM while the Act-deficient variant (Cdu1^K268E-Flag) did not (Figure 4A and B). These results suggest that the acetylase activity of Cdu1 rather than its DUB activity is required for Cdu1’s ability to stabilize InaC and IpaM proteins.

Figure 4

Download asset Open asset

The acetylase activity of Cdu1 is required to stabilize Cdu1, InaC, and IpaM.

(A) Western blot analysis of endogenous InaC and Cdu1-Flag catalytic variants expressed from a plasmid (pBOMB). HeLa cells were infected for 36 hr with WT Ct (L2 434 Bu pBOMB), a *cdu1* null strain (*cdu1*::GII pBOMB), and *cdu1* null strains expressing wild type Cdu1-Flag and the Cdu1 variants C345A-Flag (catalytic inactive), I225A-Flag (DUB deficient), and K268E-Flag (Act deficient). Cdu1-Flag variants were expressed from a pBOMB shuttle plasmid. Protein lysates from HeLa cells infected with an *inaC* null (M407) strain were used to control for the specificity of anti-InaC antibodies. Western blot images are representative of two independent experiments. (B) Western blot analysis of endogenous IpaM and Cdu1-FLAG variants in crude extracts of HeLa cells infected for 48 hr with the same strains as describe in (A). Infection of HeLa cells with *ipaM*::GII was used to test for the specificity of the anti-IpaM antibody. Western blot images are representative of two independent experiments. (C) Western blot analysis of Cdu1^C345A-Flag (catalytic inactive) and Cdu1^K268E-Flag (Act deficient) expressed in a *cdu1* null strain or WT Ct (L2 434 Bu) background after infection of HeLa cells for 24 hr. Both Cdu1 variants are stabilized by Cdu1 expressed in WT Ct. (D) Western blot analysis of Cdu1-Flag and Cdu1^C345A-Flag expressed in a *cdu1* null strain and Cdu1^C345A-Flag expressed in WT Ct (L2 434 Bu) following immunoprecipitation (anti-Flag) from HeLa cell extracts after infection for 24 hr and treatment with MG132 (25 μM, 5 hr). Western blot image is a representative blot from at least three independent experiments. (E) Western blot analysis of endogenous InaC and IpaM following immunoprecipitation of inclusion membrane enriched subcellular fractions (24hpi) with anti-acetylated lysine antibodies. Western blot image is representative of two independent experiments. (F) Western blot analysis (Flag WB) of acetylated lysine immunoprecipitates generated from inclusion membrane enriched subcellular fractions (40 hpi) derived from HeLa cells infected with WT Ct strains expressing Cdu1-Flag, CTL0480-Flag, or IpaM-Flag. Western blot image is representative of two independent experiments. (G) WB of acetylated lysine immunoprecipitates of inclusion membrane enriched fractions (24 hpi) of HeLa cells infected with WT Ct and with an *inaC* null strain (M407) expressing InaC-Flag. Western blot image is representative of two independent experiments. (H) The initiator methionine, Lys10, Lys126, and Lys263 of Cdu1 are acetylated by 24 hpi. One tyrosine (Y) residue and multiple serine (S) and threonine (T) residues in Cdu1 are also phosphorylated during Ct infection of HeLa cells (24 hpi). Three PX(S/T)P MAPK phosphorylation consensus sequence motifs were identified in the proline rich domain of Cdu1. Modified residues were identified by quantitative LC MS/MS analysis of immunoprecipitated Cdu1-Flag across three independent biological replicates. TMD: Transmembrane domain. PR: Proline rich. PSTP: PX(S/T)P motifs. Ac: Acetylation. P: Phosphorylation. Numbering corresponds to amino acids in Cdu1 protein sequence.

Figure 4—source data 1 Original western blot scans used to generate Figure 4A (anti-InaC, anti-Flag, anti-Slc1, and anti-α-tubulin).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data1-v1.zip
Download elife-87386-fig4-data1-v1.zip
Figure 4—source data 2 Original western blot scans with labeled samples and highlighted bands for Figure 4A.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data2-v1.zip
Download elife-87386-fig4-data2-v1.zip
Figure 4—source data 3 Original western blot scans used to generate Figure 4B (anti-IpaM, anti-Flag, anti-Slc1, and anti-α-tubulin).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data3-v1.zip
Download elife-87386-fig4-data3-v1.zip
Figure 4—source data 4 Original western blot scans with labeled samples and highlighted bands for Figure 4B.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data4-v1.zip
Download elife-87386-fig4-data4-v1.zip
Figure 4—source data 5 Original western blot scans used to generate Figure 4C (anti-Flag, anti-Slc1, and anti-α-tubulin).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data5-v1.zip
Download elife-87386-fig4-data5-v1.zip
Figure 4—source data 6 Original western blot scans with labeled samples and highlighted bands for Figure 4C.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data6-v1.zip
Download elife-87386-fig4-data6-v1.zip
Figure 4—source data 7 Original western blot scans used to generate Figure 4D (anti-Flag [input], anti-Slc1 [input], anti-α-tubulin [input], anti-Flag [IP:Flag], and anti-lys48 pUb [IP:Flag]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data7-v1.zip
Download elife-87386-fig4-data7-v1.zip
Figure 4—source data 8 Original western blot scans with labeled samples and highlighted bands for Figure 4D.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data8-v1.zip
Download elife-87386-fig4-data8-v1.zip
Figure 4—source data 9 Original western blot scans used to generate Figure 4E (anti-IncA [input], anti-InaC [input], anti-IpaM [input], anti-InaC [IP:Acetylated lysines], anti-IpaM [IP:Acetylated lysines], and anti-IncA [IP:Acetylated lysines]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data9-v1.zip
Download elife-87386-fig4-data9-v1.zip
Figure 4—source data 10 Original western blot scans with labeled samples and highlighted bands for Figure 4E.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data10-v1.zip
Download elife-87386-fig4-data10-v1.zip
Figure 4—source data 11 Original western blot scans used to generate Figure 4F (anti-IncA [input], anti-Flag [input], and anti-Flag [IP:Acetylated lysines]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data11-v1.zip
Download elife-87386-fig4-data11-v1.zip
Figure 4—source data 12 Original western blot scans with labeled samples and highlighted bands for Figure 4F.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data12-v1.zip
Download elife-87386-fig4-data12-v1.zip
Figure 4—source data 13 Original western blot scans used to generate Figure 4G (anti-IncA [input], anti-Flag [input], and anti-Flag [IP:Acetylated lysines]).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data13-v1.zip
Download elife-87386-fig4-data13-v1.zip
Figure 4—source data 14 Original western blot scans with labeled samples and highlighted bands for Figure 4G.: https://cdn.elifesciences.org/articles/87386/elife-87386-fig4-data14-v1.zip
Download elife-87386-fig4-data14-v1.zip

When we monitored the stability of each Flag-tagged Cdu1 variant, we observed that the catalytically inactive variant of Cdu1 (C345A-Flag) was destabilized (Figure 4A and B- Flag WB). We reasoned that Cdu1 also protects itself from being targeted for degradation in infected cells. As with InaC and IpaM, the acetylase but not the DUB activity of Cdu1 was required for Cdu1’s stability (Figure 4A and B). Although the C345A (DUB-, Act-) and K268E (Act-) amino acid substitutions in Cdu1 do not destabilize Cdu1 expressed in E. coli (Pruneda et al., 2018), it was possible that these substitutions impacted the expression and/or folding of Cdu1 in Chlamydia. To determine if these Cdu1 mutants were inherently unstable, we expressed Cdu1^C345A-Flag and Cdu1^K268E-Flag in WT L2 in the presence of endogenous Cdu1. We found that each Flag-tagged variant was stabilized (Figure 4C), indicating that endogenous Cdu1 protected the catalytically-deficient Cdu1 variants in trans. In addition, western blot analysis of immunoprecipitated Cdu1-Flag and Cdu1^C345A-Flag expressed in a cdu1::GII mutant showed that while WT Cdu1-Flag was not modified by Lys48-linked poly-ubiquitination, Cdu1^C345A-Flag was robustly modified by Lys48-linked polyUb in the presence of the proteasome inhibitor MG132 (Figure 4D). Moreover, endogenous Cdu1 protected Cdu1^C345A-Flag from Lys48-linked polyUb when Cdu1^C345A-Flag was expressed in a wild type L2 background (Figure 4D). These results indicate that the loss of Cdu1 activity likely leads to its Lys48-linked poly-ubiquitination and subsequent proteasome-dependent degradation.

Because Cdu1 autoacetylates itself and its Act activity is directed towards lysines (Pruneda et al., 2018) we postulated that Cdu1 may stabilize proteins from degradation by acetylating lysine residues that are potential targets of ubiquitination. We tested this hypothesis by assessing whether Cdu1, InaC, CTL0480, and IpaM are acetylated at lysines during infection. Fractions enriched for inclusion membranes were isolated by sub-cellular fractionation from HeLa cells infected with wild-type L2 (24 hpi), and proteins acetylated at lysines were immunoprecipitated. Western blot analysis of acetyl-lysine immunoprecipitates indicated that InaC and IpaM, but not the Inc protein IncA, were acetylated (Figure 4E). Western blot analysis of anti-acetyl-lysine immunoprecipitates of inclusion membrane-enriched membrane fractions of HeLa cells infected with L2 expressing Cdu1-Flag (24 hpi), CTL0480-Flag (40 hpi), and IpaM-Flag (40 hpi) also showed that all three Flag tagged effectors were acetylated at lysines (Figure 4F). We also determined that Flag-tagged InaC expressed in an inaC null (M407) background was acetylated at lysines as determined by western blot analysis of anti-acetyl-lysine immunoprecipitates (Figure 4G). In addition, we identified acetylated forms of Cdu1 from mass spectrometric analysis of Flag immunoprecipitates derived from extracts of HeLa cells infected with L2 expressing Cdu1-Flag, (24 hpi) (Figure 4H).

Cdu1’s acetylase activity shields inclusions from ubiquitination but is not sufficient to protect against IFNγ mediated antimicrobial activity

We reasoned that the lysine acetylase activity of Cdu1 is a prominent mechanism by which Cdu1 protects client proteins (at 24 hpi), since loss of Cdu1 or expression of the acetylase-deficient variant of Cdu1 (Cdu1^K268E-Flag) leads to a marked increase in Ub immunostaining at or near the periphery of cdu1::GII inclusions (>80% of inclusions) compared to HeLa cells infected with cdu1::GII strains complemented with wild type or DUB-deficient (I225A-Flag) Cdu1 strains (Figure 5A and B, and Figure 5—figure supplement 1A). Given that Cdu1 appears to localize exclusively at inclusion membranes, we predicted that its activity would be spatially restricted to the inclusion periphery. We tested this premise by co-infecting HeLa cells with an incA null strain (M923 (IncA^R197*), Kokes et al., 2015) and the cdu1::GII strain. IncA mediates homotypic fusion of inclusion membranes and loss of IncA results in the accumulation of multiple unfused inclusions in cells infected at high MOIs (Hackstadt et al., 1999; Suchland et al., 2000; Pannekoek et al., 2005; Figure 5C). As expected, incA mutants which retain Cdu1 activity did not accumulate Ub at or near the periphery of inclusion membranes. In HeLa cells coinfected with both cdu1::GII and M923 (IncA^R197*), cdu1::GII Cdu1^C345A and M923, or cdu1::GII Cdu1^K268E and M923, only the incA null inclusions were protected from ubiquitination (Figure 5C and D, and Figure 5—figure supplement 1B). Based on these observations, we conclude that the acetylase activity of Cdu1 protects proteins in cis and that this activity is constrained to the membrane of the pathogenic vacuole consistent with previous reports (Auer et al., 2020).

Figure 5 with 1 supplement see all

Download asset Open asset

The DUB activity of Cdu1 is not required for blocking the ubiquitination of inclusion membranes and Cdu1 is not required for protection against IFNγ mediated cellular immunity.

(A) Representative images of Ct inclusions decorated with ubiquitin during infection of HeLa cells with a *cdu1*::GII strain for 24 hr. Representative images of infected HeLa cells used for quantification of ubiquitin decorated inclusions in (B) are shown in Figure 5—figure supplement 1A. Antisera against the membrane protein Cap1 (green) was used to mark inclusion membranes. DNA stained with Hoechst is shown in blue. Scale bar: 10 μm. (B) Quantification of ubiquitinated inclusions as shown in (A). The Ub fluorescent signal was used to determine the number of infected cells with Ub decorated inclusions. The total number of ubiquitinated inclusions was divided by the total number of inclusions analyzed (defined by Cap1 staining). 87%, 86%, and 91% of inclusions were decorated with Ub in HeLa cells infected with a *cdu1* null strain and *cdu1* null strains expressing Cdu1^C345A (DUB-, Act-), and Cdu1^K268E (Act-) variants respectively. Representative images (panel (A) and Figure 5—figure supplement 1A) and quantification of ubiquitinated inclusions were obtained from inclusions imaged in 10 fields across 3 independent biological replicates for each strain. p values were determined by a student paired t-test. For cells infected with the strains WT Ct, *cdu1*::GII, and *cdu1*::GII strains expressing WT Cdu1 and the variants C345A, I225A and K268E, n (across 3 replicates) = 262, 1505, 652, 1229, 389, and 1413 respectively. Error bars depict standard deviation.(C) Representative images of HeLa cells co-infected with *cdu1*::GII (*cdu1Δ*) and *incA* null (*incA*Δ, M923) strains at 24 hpi. IncA-deficient inclusions do not fuse with other inclusions. In co-infected cells, Cdu1 present on the inclusion membranes of *incAΔ* strains did not block ubiquitination events at or near the inclusion membranes of neighboring *cdu1Δ* strains (mCherry signal from pBOMB4-MCI plasmid). Representative images of HeLa cells infected with strains quantified in (D) are shown in Figure 5—figure supplement 1B. DNA stained with Hoechst is shown in blue. Scale bar: 10 μm. (D) Quantification of *cdu1Δ* inclusions as shown in (C) in which ubiquitination events are observed in regions of *cdu1Δ* inclusion membranes that are in direct apposition to *incAΔ* inclusion membranes (junctions). The total number of *cdu1Δ* inclusions ubiquitinated at inclusion junctions was divided by the total number of inclusions analyzed (Cap1 staining). 66% and 61% of *cdu1Δ* inclusions were decorated with Ub at junctions in HeLa cells co-infected with *incAΔ* and *cdu1Δ* strains or with *incAΔ* and a *cdu1Δ* strain ectopically expressing Cdu1^K268E (Act-) respectively. Representative images (panel (C) and Figure 5—figure supplement 1B) and quantification of *cdu1Δ* inclusions ubiquitinated at junctions are derived from inclusions imaged in six fields across three independent biological replicates for each condition. p values were determined by a student paired t-test. For cells infected with the strains *cdu1*::GII and *incA* null, n (across 3 replicates) = 547 and 420 respectively. For cells co-infected with the *incA* null strain and *cdu1*::GII, or *cdu1*::GII strains expressing WT Cdu1 and the variants C345A, I225A and K268E, n (across 3 replicates) = 234, 188, 165, and 207 respectively. Error bars depict standard deviation. (E) Quantification of Ct inclusion production during infection of unprimed and IFNγ-primed (100 U/mL) A549 cells at 24 hours post infection. Inclusions were quantified by high-content imaging analysis. Plot reflects inclusion counts across nine fields of view and three independent biological replicates. Inclusion counts by each strain in unprimed A549 cells were set to 100%. Inclusion counts resulting from *cdu1*::GII and *garD*::GII strains were normalized to corresponding parental Ct inclusion (100%) counts in unprimed cells. p-values were calculated by two-way ANOVA analysis. For unprimed A549 cells infected with strains WT Ct (*garD*::GII parent), *garD*::GII, WT Ct (*cdu1*::GII parent), and *cdu1*::GII, n (across 3 replicates) = 20,212, 15,356, 10,931, and 9,535 respectively. For A549 cells pretreated with 100 U/mL IFNγ and infected with strains WT Ct (*garD*::GII parent), *garD*::GII, WT Ct (*cdu1*::GII parent), and cdu1::GII, n (across 3 replicates) = 19,082, 1,170, 7,842, and 6,368 respectively. Error bars depict standard deviation. (F) Representative images of RNF213 localizing to inclusions of WT Ct, *garD*::GII, and *cdu1*::GII strains during infection of A549 cells primed with IFNγ (100 U/mL). (G) Quantification of RNF213 localizing to Ct inclusions during infection of unprimed and IFNγ-primed (100 U/mL) A549 cells at 24 hours post infection. Plot reflects inclusion counts across six fields and three independent biological replicates. For unprimed A549 cells infected with strains WT Ct, *garD*::GII, and *cdu1*::GII, n (across 3 replicates) = 830, 817, and 620 respectively. For A549 cells pretreated with 100 U/mL IFNγ and infected with strains WT Ct, *garD*::GII, and cdu1::GII, n (across 3 replicates) = 816, 520, and 617 respectively. Error bars depict standard deviation.

Figure 5—source data 1 Excel file with numerical data for quantifying ubiquitinated inclusions in HeLa cells infected with WT CT and cdu1::GII strains, and cdu1::GII strains expressing Cdu1 C345A, I225A, and K268E variants (Figure 5B).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data1-v1.zip
Download elife-87386-fig5-data1-v1.zip
Figure 5—source data 2 Excel files with numerical data for quantifying the number of ubiquitinated inclusions in HeLa cells infected with cdu1::GII and incA null strains, and cdu1::GII strains expressing Cdu1 variants that are ubiquitinated at inclusion junctions in co-infected HeLa cells (Figure 5D).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data2-v1.zip
Download elife-87386-fig5-data2-v1.zip
Figure 5—source data 3 Excel file containing numerical data for quantifying the number of inclusions in IFN-γ-primed A549 cells infected with WT CT, garD::GII, and cdu1::GII strains (Figure 5E).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data3-v1.zip
Download elife-87386-fig5-data3-v1.zip
Figure 5—source data 4 Excel file containing numerical data for quantifying the number of RNF213-positive inclusions in IFN-γ-primed A549 cells infected with WT CT, garD::GII, and cdu1::GII strains (Figure 5G).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig5-data4-v1.zip
Download elife-87386-fig5-data4-v1.zip

Recently, the Ct inclusion membrane protein GarD was identified as a Ct effector that shields Ct from γ-interferon mediated ubiquitination by the IFNγ-inducible human ubiquitin E3 ligase RNF213 (Walsh et al., 2022). Because Cdu1 also protects the Ct inclusion from ubiquitination, we tested if Cdu1 also plays a role in protecting Ct from IFNγ-induced cell immunity. A549 cells were pretreated with IFNγ (100 U/mL) and infected with WT Ct, cdu1::GII, or garD::GII strains. Infections with a garD::GII strain led to an approximate 90% decrease in the number of inclusions formed relative to infections with its parental WT Ct strain (Figure 5E) while infections with a cdu1::GII or its parental WT Ct strain showed a modest reduction in inclusion formation (approximately 26% and 12%, respectively) (Figure 5E). These results suggest that Cdu1 likely does not play a role in protecting Ct from IFNγ-mediated cellular immunity.

We also tested whether RNF213 localizes to inclusions that lack Cdu1, as observed in IFNγ-primed A549 cells infected with garD::GII strains (Walsh et al., 2022). RNF213 did not localize to Ct inclusions when cells were infected with either WT Ct or cdu1::GII strains, regardless of whether or not the A549 cells were treated with IFNγ (Figure 5F and G). In contrast, RNF213 localized to approximately 37% of inclusions in cells infected with garD::GII mutants of unprimed A549 cells, and 81% in IFNγ-treated cells (Figure 5F and G). Based on these results, we conclude that Cdu1 does not play a role in protecting Ct from IFNγ-induced antimicrobial activity.

Cdu1 is required for F-actin assembly and Golgi ministack repositioning around the Ct inclusion, and for MYPT1 recruitment to Ct inclusions

InaC is required for Ct to assemble F-actin scaffolds and to reposition Golgi mini stacks around the periphery of the inclusion membrane (Kokes et al., 2015; Wesolowski et al., 2017; Haines et al., 2021). Because Cdu1 regulates InaC levels, we predicted that cdu1 mutants would phenocopy inaC mutants. We quantified the number of inclusions surrounded by F-actin cages at 40 hpi. In cells infected with WT L2 (parental strain of M407 (inaC null), Nguyen and Valdivia, 2012; Kokes et al., 2015), approximately 25% of inclusions were surrounded by F-actin, consistent with previous observations (Chin et al., 2012; Kokes et al., 2015; Figure 6A and B, and Figure 6—figure supplement 1). The number of inclusions surrounded by F-actin decreased to approximately 7% in cells infected with an inaC null strain (M407) and increased to approximately 49% in HeLa cells infected with an inaC null strain (M407) complemented with wild type InaC (Figure 6A and B, and Figure 6—figure supplement 1). Cells infected with cdu1::GII mutants transformed with an empty plasmid or expressing Cdu1^C345A-Flag (DUB- Act-) and Cdu1^K268E-Flag (Act-) resulted in approximately 8%, 13%, and 10% of of F-actin positive inclusions respectively (Figure 6A and B, and Figure 6—figure supplement 1). In contrast, cells infected with cdu1::GII mutants expressing Cdu1-Flag and Cdu1^I225A-Flag (DUB-) led to a marked increase in F-actin inclusions (approximately 52% and 46%, respectively) (Figure 6A and B, and Figure 6—figure supplement 1). From these observations we conclude that the acetylase activity of Cdu1 is required for Ct to promote assembly of F-actin around the Ct inclusion likely through the stabilization of InaC.

Figure 6 with 2 supplements see all

Download asset Open asset

Cdu1 is required for assembly of F-actin, Golgi ministack repositioning, and MYPT1 recruitment to the inclusion.

(A) Examples of representative images of F-actin (arrowheads) (green, Alexa Fluor Phalloidin) assembled around the Ct inclusion (magenta, anti Cdu1 and Cap1 staining) in HeLa cells infected for 40 hr. Representative images for each strain analyzed can be found in Figure 6—figure supplement 1. (B) Quantification of Ct inclusion surrounded by F-actin normalized to the total number of inclusions analyzed during infection of HeLa cells at 40 hpi. Quantification of surrounding F-actin were obtained from inclusions imaged in six fields across three independent biological replicates. p values were determined by one-way ANOVAs with a Student-Newman-Keuls post hoc test. Strains used: WT L2 (Rif-R 434 Bu, parent of M407), M407 (*inaC* null strain) p2TK2, M407 p2TK2-InaC, WT L2 (434 Bu) pBOMB, *cdu1*::GII pBOMB, *cdu1*::GII pBOMB-Cdu1 Flag, *cdu1*::GII pBOMB-Cdu1^C345A Flag, *cdu1*::GII pBOMB-Cdu1^I225A Flag, and *cdu1*::GII pBOMB-Cdu1^K268E Flag. For cells infected with the strains WT CT (parent of M407), *inaC* null, *inaC* null + InaC, WT L2, *cdu1*::GII, and *cdu1*::GII strains expressing WT Cdu1 and the variants C345A, I225A and K268E, n (across 3 replicates) = 259, 708, 780, 477, 538, 496, 370, 438, and 472 respectively. Error bars depict standard deviation.(C) Sample representative images of Golgi (anti GM130 staining, magenta) around Ct inclusions in HeLa cells infected for 24 hours. Representative images for each strain analyzed can be found in Figure 6—figure supplement 2. (D) Quantification of Golgi dispersal around the Ct inclusion during infection of HeLa cells for 24 hpi. The length of Golgi dispersed around each Ct inclusion imaged was measured and normalized to the perimeter length of each inclusion (% Golgi dispersion around the inclusion). Golgi dispersal around Ct inclusions was quantified from inclusions imaged in six fields across three independent biological replicates. p values were determined by a student paired t-test. Strains analyzed were the same ones as mentioned in (B). For cells infected with the strains WT CT (parent of M407), *inaC* null, *inaC* null + InaC, WT L2, *cdu1*::GII, and *cdu1*::GII strains expressing WT Cdu1 and the variants C345A, I225A and K268E, n (across 3 replicates) = 351, 187, 147, 169, 198, 284,187, 143, and 130 respectively. Error bars depict standard deviation. (E) Representative images of MYPT1 (magenta) at Ct inclusions (green, anti-IncA staining). Arrowheads represent *cdu1* null inclusions with low MYPT1 signal. DNA stained with Hoechst is shown in blue in panels C and E. Scale bar: 10 μm. (F) Quantification of MYPT1 at Ct inclusions as shown in (E). Representative images in (E) and quantification of MYPT1 recruitment in (F) were obtained from inclusions imaged in six fields across three independent replicates. Error bars depict standard deviation. p values were determined by a student paired t-test. For cells infected with WT Ct n (across 3 replicates) = 187 inclusions at 36 hpi and n=204 inclusions at 48 hpi. For cells infected with *cdu1*::GII n (across 3 replicates) = 202 inclusions at 36 hpi and n=168 at 48 hpi. For cells infected with *ctl0480*::GII n (across 3 replicates) = 175 inclusions at 36 hpi and n=203 inclusions at 48 hpi.

Figure 6—source data 1 Excel file containing numerical data for quantifying the number of inclusions surrounded by filamentous actin in HeLa cells infected with WT CT, inaC null, inaC null complemented with InaC, cdu1::GII, and cdu1::GII strains expressing Cdu1, Cdu1 C345A, I225A, and K268E variants (Figure 6B).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig6-data1-v1.zip
Download elife-87386-fig6-data1-v1.zip
Figure 6—source data 2 Excel files containing numerical data for quantifying the number of inclusions surrounded by dispersed Golgi mini stacks in HeLa cells infected with WT CT, inaC null, inaC null complemented with InaC, cdu1::GII, and cdu1::GII strains expressing Cdu1, Cdu1 C345A, I225A, and K268E variants (Figure 6D).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig6-data2-v1.zip
Download elife-87386-fig6-data2-v1.zip
Figure 6—source data 3 Excel files with numerical data for quantifying the localization of MYPT1 to WT and cdu1::GII inclusions (Figure 6F).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig6-data3-v1.zip
Download elife-87386-fig6-data3-v1.zip

We next quantified Golgi dispersal in infected HeLa cells at 24 hpi, a process that is also dependent on InaC (Kokes et al., 2015; Wesolowski et al., 2017). In HeLa cells infected with an inaC null strain (M407) Golgi dispersal was limited to approximately 26% of the Ct inclusion perimeter. In contrast, cells infected with either its parental WT L2 or with an inaC null strain (M407) complemented with wild type InaC, the Golgi is dispersed around 45% of the inclusion perimeter (Figure 6C and D, and Figure 6—figure supplement 2). Similarly, Golgi dispersal around inclusions during infection with WT L2 and in cdu1::GII mutants expressing wild type Cdu1-Flag or Cdu1^I225A-Flag (DUB-) was approximately 43%, 41%, and 43%, respectively (Figure 6C and D, and Figure 6—figure supplement 2). In HeLa cells infected with cdu1::GII and cdu1::GII strains expressing Cdu1^C345A-Flag (DUB- Act-), and Cdu1^K268E-Flag (Act-), Golgi repositioning was restricted to approximately 24%, 23%, and 23% of inclusion perimeters, respectively (Figure 6C and D, and Figure 6—figure supplement 2). These results confirm that both InaC and Cdu1 are required for efficient repositioning of the Golgi around the Ct inclusion as previously reported (Kokes et al., 2015; Wesolowski et al., 2017; Pruneda et al., 2018; Auer et al., 2020) and that this process is independent of Cdu1’s DUB activity but requires its acetylase activity. Moreover, our results suggest that Cdu1 promotes Golgi repositioning by protecting InaC-mediated redistribution of the Golgi around the Ct inclusion.

CTL0480 promotes recruitment of the myosin phosphatase subunit MYPT1 to the inclusion membrane where it regulates the extrusion of intact inclusions from host cells (Lutter et al., 2013; Shaw et al., 2018). Consistent with the gradual loss of CTL0480 from inclusions in cells infected with the cdu1::GII strain starting at 36 hpi (Figure 3E and Figure 3—figure supplement 1) we also observed a complete loss of MYPT1 recruitment to inclusions by 48 hpi (Figure 6E and F).

Cdu1, InaC, IpaM, and CTL0480 are required for optimal extrusion of Ct from host cells

In the absence of Cdu1, the levels of InaC, CTL0480, and IpaM decreased late in infection (36 hpi and 48 hpi, Figures 3 and 4) suggesting that a prominent role of Cdu1 is to protect these Incs from degradation late in infection. At the end of its developmental cycle, Chlamydia exits host cells by promoting cellular lysis or by extrusion of intact inclusions (Hybiske and Stephens, 2007). Ct host cell exit by extrusion is an active process requiring a remodeling of the actin cytoskeleton and the function of Inc proteins (Hybiske and Stephens, 2007; Chin et al., 2012; Lutter et al., 2013; Shaw et al., 2018; Nguyen et al., 2018). CTL0480 recruits MYPT1 (an inhibitor of Myosin II motor complexes) to the inclusion membrane which prevents premature extrusion of Ct inclusions and loss of CTL0480 leads to increased rates of extrusion by Ct from infected HeLa cells (Lutter et al., 2013; Shaw et al., 2018; Figure 7). Actin polymerization is also required for Ct extrusion (Hybiske and Stephens, 2007; Chin et al., 2012) suggesting that InaC dependent recruitment of F-actin to the inclusion may also contribute to optimal Ct extrusion. IpaM localizes to microdomains in the inclusion membrane that are proposed to function as foci for extrusion (Nguyen et al., 2018). Based on these observations, we postulated that Cdu1-mediated protection of CTL0480, InaC, and IpaM regulates the extrusion of Ct inclusions. We quantified the number of extrusions released from infected HeLa cells at 52 hpi and observed a 60% reduction in the number of extrusions in HeLa cells infected with the cdu1::GII strain relative to cells infected with WT L2 (Figure 7A and B). Complementation of cdu1::GII with either wild type Cdu1-Flag or Cdu1^I225A-Flag (DUB-) restored extrusion production to near wild type levels. In contrast, HeLa cells infected with cdu1::GII mutants expressing Cdu1^K268E-Flag (Act-), or inaC (inaC::GII, Wesolowski et al., 2017) and ipaM (ipaM::GII, Meier et al., 2023) null strains led to a 42%, 75%, and 58% reduction in extrusion production respectively (Figure 7A and B). The decrease in the number of extruded inclusions by these strains was not attributed to defects in inclusion biogenesis as they produced a comparable number of inclusions at 48 hpi relative to cells infected with WT L2 (Figure 7—figure supplement 1). Consequently, we infer that InaC, IpaM, and Cdu1 collectively contribute to the promotion of optimal extrusion of Ct inclusions from host cells, with Cdu1 playing a central regulatory role by protecting these effectors from degradation.

Figure 7 with 1 supplement see all

Download asset Open asset

Cdu1, InaC, and IpaM are required for optimal extrusion of Ct inclusions from HeLa cells.

(A) Representative images of extrusions isolated from HeLa cell monolayers infected with Ct strains for 52 hr. Scale bar: 200 μm (B) Quantification of the number of extruded inclusions produced by infected HeLa cell monolayers. p values were determined by a student paired t-test. (C) Quantification of the size of extruded inclusions quantified in (B). Extruded inclusions varied in size among cells infected with wild type L2 (average:43 μm), *ipaM* mutants (average: 56 μm) and *cdu1* mutants complemented wild type Cdu1 (average: 52 μm) and Cdu1^I225A (DUB-) (average: 60 μm). p values were determined by one-way ANOVAs with a Student-Newman-Keuls post hoc test. Representative images in (A) and quantification of extrusion number in (B) and extrusion size in (C) are based on images obtained from six fields across three independent biological replicates. For extrusions isolated from Hela cells infected with WT CT, *cdu1*::GII, *inaC*::GII, *ipaM*::GII, *ctl0480*::GII, and *cdu1*::GII strains expressing WT Cdu1 and the variants I225A and K268E, n (across 3 replicates) = 2,580, 1,025, 637, 1,194, 3,726, 2,321, 2,198, and 1,091 respectively. Error bars depict standard deviation.

Figure 7—source data 1 Excel files containing numerical data for quantifying the number of extrusions produced and the size of extrusions in WT CT, cdu1::GII strains, and cdu1::GII strains expressing Cdu1, I225A, and K268E variants from infected HeLa cells (Figure 7B and C).: https://cdn.elifesciences.org/articles/87386/elife-87386-fig7-data1-v1.zip
Download elife-87386-fig7-data1-v1.zip

In contrast, infection of HeLa cells with a ctl0480::GII mutant strain led to an increase in the number of extruded inclusions as previously observed (Shaw et al., 2018; Figure 7A and B). Therefore, even though the Cdu1-mediated protection of InaC and IpaM is important for the extrusion of inclusions and cdu1 mutants phenocopy the loss of InaC and IpaM, the phenotypic similarities do not extend to the increased number of extruded inclusions observed in cells infected with the ctl0480::GII mutant strain (Figure 7A and B). We infer from these observations that functions for both InaC and IpaM in the extrusion of inclusions are epistatic to CTL0480. Extruded inclusions produced during infection of HeLa cells also varied in size with an average diameter of 40 μm (Figure 7A and C). Interestingly, the loss of IpaM and over expression of Cdu1-Flag and Cdu1^I225A-Flag (DUB-) shifted the size distribution of extrusions toward larger extrusions (Figure 7A and C) suggesting that Ct regulates the size of extruded inclusions through Cdu1.

Discussion

Several Chlamydia Inc proteins regulate interactions between the pathogenic vacuole and the host cytoskeleton, organelles, and vesicular trafficking pathways. These Inc proteins also modulate host cell death programs and promote Chlamydia exit from host cells (reviewed in Bugalhão and Mota, 2019). Given the central roles that Incs play in promoting Chlamydia intracellular infection, it is not surprising that they are targeted for inactivation by host cellular defenses. In response, Chlamydia has evolved mechanisms to protect Incs. In this study, we show that the acetylase activity of the effector Cdu1 protects itself and three Inc proteins; InaC, IpaM, and CTL0480, from ubiquitination and degradation (Figure 8). Interestingly, all three Inc proteins play prominent roles in regulating the extrusion of inclusions from host cells (Figure 7). Observations that the encapsulation of Chlamydia within an extruded inclusion enhances survival of Chlamydia within macrophages (Zuck et al., 2017) together with the broad conservation of extrusion as an exit strategy among Chlamydia (Zuck et al., 2016) suggests that this mechanism is important for Chlamydia pathogenesis. Notably, a cdu1 mutant strain (cdu1:: Tn, Fischer et al., 2017) displays reduced bacterial loads in a murine model of upper genital tract infections (Fischer et al., 2017). While neither this strain nor our cdu1::GII mutant strain shows evident growth impairments during infection of HeLa cells (data not shown), the observed reduction in bacterial load in the absence of Cdu1 in animal models of infection could potentially stem from defects in extrusion production or from perturbations in Cdu1-dependent regulation of extrusion size. Thus, targeting Inc proteins that regulate extrusion for Ub-mediated destruction may be advantageous for the host. For instance, targeting InaC for degradation would limit F-actin dependent extrusions (Hybiske and Stephens, 2007; Chin et al., 2012) and InaC-dependent microtubule scaffolds around the inclusion (Wesolowski et al., 2017; Haines et al., 2021). CTL0480 functions as an inhibitor of extrusions through its role in modulating the activity of myosin light chain 2 (MLC₂) (Lutter et al., 2013; Shaw et al., 2018). IpaM localizes to specialized microdomains in the inclusion membrane which are also sites of enrichment for over 9 inclusion membrane proteins including Ctl0480 and MrcA, both of which are required for Chlamydia extrusion (Mital et al., 2010; Lutter et al., 2013; Nguyen et al., 2018). We also find that the loss of IpaM shifted the size distribution of extrusions towards larger inclusions (Figure 7). We speculate that heterogeneity in the size of extrusions might facilitate uptake of some extrusions by innate immune cells at infected mucosal sites to promote Chlamydia LGV dissemination to distal sites in the genital tract and avoid clearance of Chlamydia by other immune cells (Zuck et al., 2017).

Figure 8

Download asset Open asset

A model for acetylation mediated protection of the Inc proteins InaC, IpaM, and Ctl0480 from degradation.

The cellular Ub machinery targets *C. trachomatis* effectors, including the Inc proteins InaC, IpaM, and CTL0480 for ubiquitination and subsequent protein degradation. *C. trachomatis* counters such defense mechanisms by translocating Cdu1 which protect itself and all three Inc proteins from being targeted for ubiquitination and degradation through its acetylase activity. Cdu1 protects InaC and enables the recruitment of F-actin scaffolds and Golgi ministacks to the inclusion perimeter and CTL0480 to recruit the Myosin II regulator MYPT1. All three inclusion proteins and Cdu1 promote extrusion and dissemination of *C. trachomatis* inclusions late in infection.

© 2024, Bastidas et al. Figure 8 was created using BioRender, and is published under a CC BY-NC-ND 4.0 license. Further reproductions must adhere to the terms of this license.

Effectors that modulate the activity of other translocated effectors are referred to as ‘metaeffectors’, a term coined by Kubori and colleagues after observing that the L. pneumophila effector LubX which functions as an E3 ligase, ubiquitinates the translocated effector SidH leading to its degradation (Kubori et al., 2010). Several other effector-metaeffector interactions have been described in L. pneumophila, Salmonella enterica, and Brucella abortus which regulate the activity of other effectors either directly or indirectly by modifying the same host target or cellular process (Kubori et al., 2010; Neunuebel et al., 2011; Jeong et al., 2015; Urbanus et al., 2016; Smith et al., 2020; Iyer and Das, 2021). In this context, we propose that Cdu1 functions as a metaeffector in Ct to protect multiple effectors. We also observed that Cdu1 interactions with InaC, IpaM, and CTL0480 likely occur independently from each other and that the kinetics of degradation in the absence of Cdu1 varies for each Inc (Figure 3).

Our findings indicate that the DUB activity of Cdu1 was not required to protect InaC, IpaM, and CTL0480 from ubiquitination. Instead we find that Cdu1’s lysine acetylase activity is required to protect these Inc proteins and Cdu1 itself from ubiquitination. Indeed, we found that all three Incs and Cdu1 are acetylated at lysines in infected cells. However, we were unable to determine if lysine acetylation in all four proteins was dependent on Cdu1s’ Act activity or if these PTMs are protective. Why the DUB activity of Cdu1 is unable to compensate for loss of its Act activity remains unknown. It is possible that Cdu1, like other DUBs, is regulated by PTMs (Komander et al., 2009a). For instance, phosphorylation of human CYLD inhibits its DUB activity towards TRAF2 while phosphorylation of human USP8 inhibits its DUB activity toward EGFR (Reiley et al., 2005; Mizuno et al., 2007). Mass spectrometry analysis of immunoprecipitated Flag tagged Cdu1 expressed in Ct revealed that Cdu1 is phosphorylated at multiple serine and threonine residues (Figure 4H) as previously suggested (Zadora et al., 2019). We identified three PX(S/T)P MAPK phosphorylation consensus sequence motifs in the proline rich domain (PRD) of Cdu1, suggesting that MAPKs may regulate the DUB activity of Cdu1.

Cdu1 homologs are found in multiple Chlamydia species including C. trachomatis, C. muridarum, C. suis, C. psitacci, C. abortus, C. caviae, and C. felis but is notably absent in the genomes of C. pneumoniae and C. pecorum. The acquisition of a second deubiquitinase paralog (Cdu2) has also occurred in C. trachomatis, C. muridarum, and C. suis. In the genomes of all three species, cdu2 resides directly adjacent to cdu1; an arrangement that presumptively arose from a gene duplication event. Cdu2 is a dedicated ULP with deubiquitinating and deneddylating activities (Misaghi et al., 2006; Pruneda et al., 2016). New evidence suggest that both paralogues might not be functionally redundant. The crystal structure of Cdu2 has revealed differences in residues involved in substrate recognition between Cdu1 and Cdu2 and that each paralog might recognize polyUb chains differently (Hausman et al., 2020). The processivity rates for removal of terminal Ub from polyUb chains also differs between both isopeptidases with Cdu2 exhibiting limited trimming of polyUb as compared to Cdu1 (Hausman et al., 2020). Moreover, Cdu2 lacks the proline rich domain found in Cdu1 which might be important for regulation of Cdu1 enzymatic activity. The presence of Cdu2 might also explain the low incidence of human and Ct proteins that were differentially ubiquitinated in the absence of Cdu1 (Figure 1). Whereas several Chlamydia species have acquired either one or two deubiquitinase paralogs, both C. pneumoniae and C. pecorum have not. Instead, both species have acquired an unrelated deubiquitinase (ChlaOTU) belonging to the OTU family of proteases (Makarova et al., 2000; Furtado et al., 2013). Curiously, ChlaOTU is also found in C. psitacci, C. abortus, C. caviae, and C. felis all of which encode only Cdu1 and is absent in C. trachomatis, C. muridarum, and C. suis, all of which encode Cdu1 and Cdu2. It is noteworthy that Chlamydia species have independently acquired deubiquitinases multiple times (Cdu1, Cdu2, ChlaOTU) and that some of these deubiquitinases have evolved into moonlighting enzymes reflecting the diverse strategies adopted by pathogenic Chlamydia as they adapt to their particular niche.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Chlamydia trachomatis L2 434 Bu)	cdu1	BV-BRC (Bacterial and Viral Bioinformatics resource center); Refseq (NCBI Reference Sequence Database)	fig\|471472.4.peg.264 (BV-BRC) CTL0247 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	inaC	BV-BRC; Refseq	fig\|471472.4.peg.199 (BV-BRC) CTL0184 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	ipaM	BV-BRC; Refseq	fig\|471472.4.peg.511 (BV-BRC) CTL0476 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	CTL0480	BV-BRC; Refseq	fig\|471472.4.peg.516 (BV-BRC) CTL0480 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	cpoS	BV-BRC; Refseq	fig\|471472.4.peg.517 (BV-BRC) CTL0481 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	incA	BV-BRC; Refseq	fig\|471472.4.peg.406 (BV-BRC) CTL0374 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	slc1	BV-BRC; Refseq	fig\|471472.4.peg.323 (BV-BRC) CTL0299 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	rpoB	BV-BRC; Refseq	fig\|471472.4.peg.608 (BV-BRC) CTL0567 (Refseq)
Gene (Chlamydia trachomatis L2 434 Bu)	garD	BV-BRC; Refseq	fig\|471472.4.peg.421 (BV-BRC) CTL0390 (Refseq)
Gene (Homo sapiens)	MYPT1	NCBI	PPP1R12A
Cell line (Homo sapiens)	Hela	ATCC	Cat# CCL-2 RRID:CVCL_0030
Cell line (Cercopithecus aethiops)	Vero	ATCC	Cat# CCL-81 RRID:CVCL_0059
Cell line (Homo sapiens)	HEK 293T	ATCC	Cat# CRL-3216 RRID:CVCL_0063
Cell line (Homo sapiens)	A549	ATCC	Cat# CCL-185 RRID:CVCL_0023
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pOPIN-GFP_Cdu1 FL (aa 1–401)	Jonathan Pruneda (Oregon Health and Science University)	N/A	Used for Cdu1 co-immunoprecipitations in transfected cells
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pOPIN-GFP_Cdu1 TMD- (aa 71–401)	Jonathan Pruneda (Oregon Health and Science University)	N/A	Used for Cdu1 co-immunoprecipitations in transfected cells
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pOPIN-GFP_Cdu1 CD- (aa 1–130)	Jonathan Pruneda (Oregon Health and Science University)	N/A	Used for Cdu1 co-immunoprecipitations in transfected cells
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pCDNA-DEST53 (w/o GFP)_InaC (CT813)–3XFLAG	This paper	N/A	Construct for showing Cdu1-InaC co-IP in transfected cells
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pcDNA3.1/nV5-DEST_IpaM	This paper	N/A	Construct for showing Cdu1-IpaM co-IP in transfected cells
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pcDNA3.1/nV5-DEST_CTL0480	This paper	N/A	Construct for showing Cdu1-CTL0480 co-IP in transfected cells
Transfected construct (Chlamydia trachomatis L2 43 Bu)	pcDNA3.1/nV5-DEST_CpoS	This paper	N/A	Construct for showing lack of Cdu1-Cpos co-IP in transfected cells
Biological sample (Chlamydia trachomatis L2 434 Bu)	Chlamydia trachomatis LGV biovar L2 434 Bu (L2)	Richard Stephens (UC Berkeley)	N/A	Reference Chlamydia trachomatis (L2) strain
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 pBOMB-MCI (Parent: LGV L2 434 Bu)	This paper	N/A	L2 strain transformed with empty expression plasmid
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 pBOMB-MCI_CTL0480-3XFlag (Parent: LGV L2 434 Bu)	This paper	N/A	L2 strain transformed with plasmid expressing CTL0480-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 pBOMB-MCI_IpaM-3XFlag (Parent: LGV L2 434 Bu)	This paper	N/A	L2 strain transformed with plasmid expressing IpaM-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 pBOMB-MCI_Cdu1-3XFlag (Parent: LGV L2 434 Bu)	This paper	N/A	L2 strain transformed with plasmid expressing Cdu1-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 pBOMB-MCI_Cdu1^C345A-3XFlag (Parent: LGV L2 434 Bu)	This paper	N/A	L2 strain transformed with plasmid expressing Cdu1^C345A –3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 pBOMB-MCI_Cdu1^K268E-3XFlag (Parent: LGV L2 434 Bu)	This paper	N/A	L2 strain transformed with plasmid expressing Cdu1^K268E –3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 cdu1::GII aadA (Parent: LGV L2 434 Bu)	This paper	N/A	L2 cdu1 null strain (Spec^R)
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 cdu1::GII aadA pBOMB-MCI (Parent: L2 cdu1::GII aadA)	This paper	N/A	L2 cdu1 null strain transformed with empty expression plasmid
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 cdu1::GII aadA pBOMB-MCI_Cdu1-3XFlag (Parent: L2 cdu1::GII aadA)	This paper	N/A	L2 cdu1 null strain transformed with plasmid expressing WT Cdu1-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 cdu1::GII aadA pBOMB-MCI_Cdu1^C345A-3XFlag (Parent: L2 cdu1::GII aadA)	This paper	N/A	L2 cdu1 null strain transformed with plasmid expressing WT Cdu1^C345A-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 cdu1::GII aadA pBOMB-MCI_Cdu1^I225A-3XFlag (Parent: L2 cdu1::GII aadA)	This paper	N/A	L2 cdu1 null strain transformed with plasmid expressing WT Cdu1^I225A-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 cdu1::GII aadA pBOMB-MCI_Cdu1^K268E-3XFlag (Parent: L2 cdu1::GII aadA)	This paper	N/A	L2 cdu1 null strain transformed with plasmid expressing WT Cdu1^K268E-3XFlag
Biological sample (Chlamydia trachomatis L2 434 Bu)	L2 Rif-R (Parent: L2 434 Bu)	PMID:22232666	N/A	Rifampin resistant L2 strain (L2 434 Bu)
Biological sample (Chlamydia trachomatis L2 434 Bu)	M407 (inaC C307T, InaC Q103*) (Parent: L2 Rif-R)	PMID:25920978	N/A	L2 strain bearing a nonsense mutation in inaC (inaC null srain)
Biological sample (Chlamydia trachomatis L2 434 Bu)	M407 p2TK2 (Parent: M407)	PMID:25920978	N/A	L2 inaC null strain transformed with empty expression plasmid
Biological sample (Chlamydia trachomatis L2 434 Bu)	M407 p2TK2_InaC (Parent: M407)	PMID:25920978	N/A	L2 inaC null strain transformed with plasmid expressing WT InaC (untagged)
Biological sample (Chlamydia trachomatis L2 434 Bu)	M407 p2TK2_InaC-3X Flag (Parent: M407)	This paper	N/A	L2 inaC null strain transformed with plasmid expressing WT InaC-3X Flag
Biological sample (Chlamydia trachomatis L2 434 Bu)	inaC::GII bla (Parent: L2 434 Bu)	PMID:28465429	N/A	L2 inaC null strain (Penicillin^R)

Share this article

Cite this article

The C. trachomatis inclusion membrane proteins InaC, IpaM, and CTL0480 are ubiquitinated in the absence of Cdu1.

Figure 1—source data 1

Cdu1 associates with InaC, IpaM, and CTL0480.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

Figure 2—source data 4

Figure 2—source data 5

Figure 2—source data 6

Figure 2—source data 7

Figure 2—source data 8

Figure 2—source data 9

Cdu1 stabilizes InaC, IpaM, and CTL0480.

Figure 3—source data 1

Figure 3—source data 2

Figure 3—source data 3

Figure 3—source data 4

Figure 3—source data 5

Figure 3—source data 6

The acetylase activity of Cdu1 is required to stabilize Cdu1, InaC, and IpaM.

Figure 4—source data 1

Figure 4—source data 2

Figure 4—source data 3

Figure 4—source data 4

Figure 4—source data 5

Figure 4—source data 6

Figure 4—source data 7

Figure 4—source data 8

Figure 4—source data 9

Figure 4—source data 10

Figure 4—source data 11

Figure 4—source data 12

Figure 4—source data 13

Figure 4—source data 14

The DUB activity of Cdu1 is not required for blocking the ubiquitination of inclusion membranes and Cdu1 is not required for protection against IFNγ mediated cellular immunity.

Figure 5—source data 1

Figure 5—source data 2

Figure 5—source data 3

Figure 5—source data 4

Cdu1 is required for assembly of F-actin, Golgi ministack repositioning, and MYPT1 recruitment to the inclusion.

Figure 6—source data 1

Figure 6—source data 2

Figure 6—source data 3

Cdu1, InaC, and IpaM are required for optimal extrusion of Ct inclusions from HeLa cells.

Figure 7—source data 1

A model for acetylation mediated protection of the Inc proteins InaC, IpaM, and Ctl0480 from degradation.

Author details

Robert J Bastidas

Contribution

For correspondence

Competing interests

Mateusz Kędzior

Contribution

Competing interests

Robert K Davidson

Contribution

Contributed equally with

Competing interests

Stephen C Walsh

Contribution

Contributed equally with

Competing interests

Lee Dolat

Contribution

Competing interests

Barbara S Sixt

Contribution

Competing interests

Jonathan N Pruneda

Contribution

Competing interests

Jorn Coers

Contribution

Competing interests

Raphael H Valdivia

Contribution

Competing interests

Downloads (link to download the article as PDF)