The success of Mycobacterium tuberculosis (Mtb) as a human pathogen results from its ability to avoid, resist, or subvert immunological mechanisms that are effective against most pathogens. Mtb has a remarkable ability to withstand the microbicidal arsenal of host macrophages, including the low pH of the lysosome and a diverse array of toxic reactive oxygen species (ROS) produced by the host respiratory burst. However, relatively little is known about the molecular mechanisms of resistance to these toxic insults.

Upon phagocytosis of a pathogen, macrophages and neutrophils unleash a potent microbicidal arsenal, including a respiratory burst that leads to the rapid release of ROS that kill and degrade most microbes (El-Benna et al., 2009; Nathan, 2006; Segal, 2005). The production of ROS is initiated when NADPH oxidase assembles on pathogen-containing phagosomes and initiates a series of reactions that produce superoxide, hydrogen peroxide, hyperchlorous acid, lipid peroxides, and other toxic reactive molecules. Importantly, Mtb is highly resistant to oxidative stress, enabling the bacterium to survive within macrophages and neutrophils and establish chronic infection (Chan et al., 1992). Although some oxidative defense mechanisms have been characterized (Ehrt and Schnappinger, 2009; Nambi et al., 2015; Voskuil et al., 2011), the Mtb genome encodes a large number of enzymes that could participate in defense against oxidative stress that remain uncharacterized. For example, Mtb encodes ~15 peroxidases, a class of enzymes with diverse functions that form the front line of oxidative defense in bacteria (Mishra and Imlay, 2012; Singh and Eltis, 2015). It is unclear why Mtb encodes such a diversity of peroxidases, and little is known about what individual roles these enzymes may play in both resistance to oxidative stress and in normal bacterial physiology. One possibility is that specific peroxidase genes may participate in defense against different types of ROS generated during the respiratory burst. One of the most intriguing peroxidases encoded by Mtb is DyP, a member of the dye-decolorizing peroxidase family of proteins. DyP enzymes are unique amongst peroxidases in that some family members have been shown to have optimal activity at pH 4–5 (Brown et al., 2012; Singh and Eltis, 2015; Uchida et al., 2015). Furthermore, DyP is the only Mtb peroxidase predicted to be packaged inside a proteinaceous organelle known as a bacterial nanocompartment (Contreras et al., 2014).

Bacterial cells were long thought to lack compartmentalization of function. However, the identification of organelle-like structures including microcompartments, anammoxosomes, magnetosomes, and, most recently, encapsulin nanocompartments has revolutionized our understanding of bacterial cell biology (Ferousi et al., 2017; Kerfeld et al., 2018; Uebe and Schüler, 2016). Characterized nanocompartments are proteinaceous shells that are 24–45 nm in diameter and comprised of 60–240 subunits of a single protomer (Giessen et al., 2019; Nichols et al., 2017). This shell surrounds (‘encapsulates’) an enzymatic cargo protein (Nichols et al., 2017). Although putative encapsulin systems have been identified in >900 bacterial and archaeal genomes (Giessen and Silver, 2017; Nichols et al., 2020), very little is known about their physiological function. Based on genomic organization, encapsulin systems are often predicted to compartmentalize enzymes involved in oxidative stress defense, iron storage, and anaerobic ammonium oxidation (Giessen and Silver, 2017). However, the function of an encapsulin system has only ever been demonstrated in a single bacterial species, Myxococcus xanthus (McHugh et al., 2014), and the physiological role of nanocompartments is largely unexplored.

Here, we demonstrate that an Mtb nanocompartment containing DyP is crucial for resisting oxidative stress at low pH. Mutants lacking the nanocompartment are highly attenuated when exposed to H₂O₂ at pH 4.5, the pH of the lysosome. We show that this attenuation is linked to fatty acid metabolism in the bacteria. Further, we show that encapsulation of the DyP enzyme promotes its function, the first report to demonstrate a role for encapsulation in endogenous bacterial physiology. Finally, we show that the Mtb nanocompartment system is required for full growth in macrophages, and for resistance to the antibiotic pyrazinamide (PZA), demonstrating the importance of this system for a globally significant pathogen.

The Mtb genome encodes the predicted encapsulin gene Rv0798c/Cfp29 (Contreras et al., 2014) in a two-gene operon with Rv0799c, the dye-decolorizing peroxidase DyP (Figure 1A). Overexpression of the predicted Mtb encapsulin gene in Escherichia coli was previously shown to result in the formation of nanocompartment-like structures (Contreras et al., 2014). Three potential cargo proteins for the nanocompartment were proposed based on a putative shared encapsulation targeting sequence: DyP, FolB, and BrfB. In E. coli, overexpression of each protein with Cfp29 resulted in encapsulation. However, this study did not address whether Mtb produces endogenous nanocompartments or identify the specific function of these compartments in Mtb biology. A transposon screen has identified Cfp29 as a gene required for growth in mice (Zhang et al., 2013) and Cfp29 has long been known as an immunodominant T cell antigen in both mice and human TB patients (Weldingh and Andersen, 1999). Taken together, these results suggest that Mtb may produce an encapsulin nanocompartment that is important for pathogenesis.

Figure 1 with 1 supplement see all

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To confirm the previous finding that heterologous expression of Rv0798c and Rv0799c in a host species results in the assembly of an encapsulin system, we expressed these genes in E. coli and isolated nanocompartments. Clarified protein lysates from E. coli were purified by ultracentrifugation and size-exclusion chromatography. Assembled encapsulin nanocompartments are distinguishable by their high molecular weight (Nichols et al., 2017). Indeed, a fraction from the purification contained a high molecular weight species > 260 kDa observable on an SDS-PAGE gel (Figure 1—figure supplement 1A). Fractions containing putative nanocompartments were pooled and imaged using transmission electron microscopy, which revealed the presence of icosahedral structures with the expected diameter of ~25 nm (Figure 1B and C).

To determine whether Mtb produces nanocompartments under normal laboratory growth conditions, we performed an ultracentrifugation-based nanocompartment isolation strategy using wild-type H37Rv strain bacteria grown to mid-log phase (Figure 1—figure supplement 1B). Mass spectrometry analysis of the nanocompartment fraction identified both the encapsulin protein Cfp29 and the peroxidase DyP (Figure 1D). TEM analysis confirmed the presence of nanocompartment particles ~25 nm in diameter (Figure 1E). Interestingly, Rv1762c, a protein of unknown function, was consistently identified in purified nanocompartment preparations from Mtb (Figure 1D). We were unable to identify either FolB or BrfB in nanocompartments from Mtb, suggesting that these proteins are not endogenous substrates for encapsulation under normal laboratory growth conditions for Mtb.

Cfp29 (‘culture filtrate protein 29’) was originally identified in the supernatants of Mtb cells grown in axenic culture (Rosenkrands et al., 1998). As Cfp29 lacks a secretory signal sequence and is part of a large macromolecular complex, it is unclear how a nanocompartment could be actively secreted. However, nanocompartment structures are remarkably stable and it is possible that nanocompartments released from dying bacteria accumulate in culture as they are highly resistant to proteolysis/degradation (Cassidy-Amstutz et al., 2016; Nichols et al., 2017).

DyP proteins are known to have low pH optima (Contreras et al., 2014; Uchida et al., 2015). To demonstrate that Mtb DyP has a similarly low pH optimum, SUMO-tagged unencapsulated DyP was purified from E. coli (Figure 1—figure supplement 1C), and the peroxidase activity was evaluated at a range of pH values using the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) dye-decolorizing assay previously used to characterize DyP proteins (Ahmad et al., 2011; Contreras et al., 2014). Similar to the Vibrio cholerae DyP (Uchida et al., 2015), purified Mtb DyP had increased enzymatic activity in low pH environments, with the greatest efficacy at pH 4.0, the lowest pH tested (Figure 1F). We next tested the ability of encapsulated DyP to degrade ABTS across a range of pH values. Similar to free DyP, the encapsulated enzyme had the highest activity at pH ~4.0 (Figure 1G). Taken together, these data demonstrate the stability and functionality of Mtb nanocompartments under acid stress, a condition that mimics the host lysosomal environment. The Mtb DyP protein was previously shown to function as a bona fide dye-decolorizing peroxidase (Contreras et al., 2014). Because peroxidases consume H₂O₂, they often participate in defense against oxidative stress (Mishra and Imlay, 2012). During macrophage infection, the phagocytes initiate an oxidative burst that exposes Mtb to H₂O₂ (El-Benna et al., 2009). We therefore reasoned that DyP-containing nanocompartments may function to protect Mtb from H₂O₂-induced stress. To test this hypothesis, we created a mutant strain lacking both genes from the nanocompartment operon (Figure 1A, Δoperon). Lysates from Δoperon mutants were used for nanocompartment purification (data not shown) and western blot analysis using an antibody for Cfp29 as a probe (Figure 1—figure supplement 1D). Data from these analyses revealed that Δoperon mutants cannot produce viable nanocompartments. In addition, we isolated a transposon mutant (DyP::Tn) containing an insertion in DyP, a mutation that eliminated expression of both Cfp29 and DyP (Figure 1—figure supplement 1E).

To test whether DyP-containing nanocompartments are required for defense against H₂O₂, we exposed wild-type and DyP::Tn Mtb to increasing concentrations of H₂O₂ and monitored bacterial survival. Mutants lacking DyP nanocompartments were more susceptible to H₂O₂ when compared with wild-type bacteria as measured by OD₆₀₀ (Figure 2A) and by plating for colony-forming units (CFU) (Figure 2B). However, the phenotype was relatively modest. We next reasoned that DyP nanocompartments might be required to resist oxidative stress in acidic environments that more closely mimic the in vivo environment. During infection, Mtb bacilli encounter the low pH of the phagolysosome and the ability to tolerate low pH is required for Mtb survival in both infected macrophages and mice (Vandal et al., 2008). We therefore tested the susceptibility of DyP mutants to a combination of H₂O₂ and acid stress (pH 4.5). Wild-type Mtb was able to withstand these conditions and did not significantly decrease in number over a 3-day exposure period (Figure 2C). In contrast, the Δoperon mutant was resistant to each stressor individually, but was highly susceptible to H₂O₂ at pH 4.5 (Figure 2C). Thus, DyP-containing nanocompartments are required to protect bacteria from oxidative stress at low pH.

Figure 2

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We next sought to determine whether encapsulation of DyP is important for its activity in bacterial cells. To accomplish this, DyP::Tn mutants were complemented with constructs expressing DyP alone (pDyP), Cfp29 encapsulin alone (pCfp29), or both proteins (pOperon). As expected, expression of DyP did not restore production of nanocompartments, whereas expression of Cfp29 alone resulted in the formation of empty nanocompartment structures (Figure 1—figure supplement 1E). Coexpression of both proteins resulted in the formation of DyP-containing nanocompartments in the DyP::Tn mutant (Figure 1—figure supplement 1E). We exposed the full set of complemented mutants to H₂O₂ at pH 4.5 and determined survival after 3 days by plating for CFU. As expected, DyP::Tn was attenuated for survival under these conditions when compared to wild-type bacteria (Figure 2D). Restoring expression of the nanocompartment shell protein in the absence of DyP had no effect on bacterial survival (Figure 2D). Complementation by overexpression of unencapsulated DyP was sufficient to confer almost wild-type levels of resistance to oxidative and acid stress (Figure 2D). However, complementation with both Cfp29 and DyP resulted in enhanced resistance to these stressors when compared to mutants complemented with DyP alone (Figure 2D), suggesting that encapsulation of DyP enhances its function.

In the mutant complemented with DyP alone, it is possible that overexpression of the peroxidase compensates for the lack of encapsulation. We therefore examined the importance of DyP encapsulation in mutant strains in which we deleted Cfp29 only (Δcfp29). Deletion of Cfp29 resulted in an ~0.5 log decrease in bacterial viability after 3 days of exposure to H₂O₂ at pH 4.5, confirming that encapsulation of the peroxidase in the shell protein is important for full protection (Figure 2E). As expected, greater attenuation was observed in the mutant lacking both DyP and Cfp29 (Δoperon). Complementation of the Δcfp29 mutant fully restored resistance (Figure 2F). Taken together, these data demonstrate that encapsulation of DyP has a modest, yet significant impact on protection against oxidative stress in low pH conditions.

We next sought to test whether nanocompartment mutants have altered redox homeostasis in the presence of oxidative stress. We transformed wild-type and mutant strains with Mrx1-roGFP, a fluorescent reporter of redox potential in mycobacteria (Bhaskar et al., 2014). Remarkably, the Δoperon mutant had an intracellular environment at baseline that was more oxidizing than wild-type bacteria exposed to H₂O₂ (Figure 2G), indicating a failure of redox homeostasis in mutants lacking the DyP encapsulin system. This oxidizing cellular environment was further enhanced by treatment with H₂O₂ (Figure 2G). We did not observe a difference in the redox state of Δcfp29. Thus, mutants lacking DyP-containing nanocompartments exhibit significant dysregulation of redox homeostasis.

The Mtb genome encodes ~15 putative peroxidase genes, including DyP. To determine whether other bacterial peroxidases participate in the defense against oxidative stress at low pH, we performed a transposon sequencing (Tn-seq) screen (Griffin et al., 2012; van Opijnen and Camilli, 2013). To do so, we created a transposon library containing ~100K individual transposon mutants and exposed this library to H₂O₂ at pH 4.5 for 3 days, at which point the surviving bacteria were plated. Transposon gene junctions were amplified and sequenced from the recovered bacteria, and the sequencing data were analyzed using TRANSIT (DeJesus et al., 2015; Supplementary file 1). These data revealed that of the 18 putative enzymes encoded by Mtb that could be involved in oxidative defense, including peroxidases, catalases, and superoxide dismutases, only two genes were required for survival in the culture conditions—DyP and KatG (Figure 3A). KatG encodes a catalase-peroxidase that is important for defense against oxidative stress in the host (Li et al., 1998; Pym et al., 2002). Coincidentally, KatG is also required for activation of isoniazid (INH), a central component of anti-TB therapy. Approximately 10% of TB cases are caused by INH-resistant bacteria, many of which have loss-of-function mutations in KatG (Seifert et al., 2015). Importantly, KatG variants that do not activate INH often display a concomitant decrease in peroxidase and catalase activity (DeVito and Morris, 2003; Mo et al., 2004; Wei et al., 2003). Both the high proportion of KatG mutant bacteria in the human population and studies of virulence in animals suggest that KatG mutants are still capable of growth in vivo (Nieto et al., 2016; Pym et al., 2002). The fact that both DyP and KatG mutants are important for survival in oxidative stress at low pH suggests that the DyP nanocompartment may provide redundancy that compensates for a loss of KatG in INH-resistant strains of Mtb.

Figure 3

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In the Tn-seq data, we observed that mutants involved in lipid or cholesterol metabolism were also attenuated when exposed to H₂O₂ at pH 4.5 (Figure 3B). Mtb biology is highly linked to lipid biology; Mtb granulomas are rich in free fatty acids (Kim et al., 2010) and the bacteria utilize lipids as a source of nutrition both during macrophage infection ex vivo and growth in vivo (Marrero et al., 2010; Russell, 2011; Russell et al., 2019). In particular, mutants containing transposon insertions in three independent subunits of the Mce1 complex, which is required for import of fatty acids (Nazarova et al., 2019), were susceptible to oxidative stress under acidic conditions (Figure 3B). The standard Mtb culture medium, 7H9, contains bovine serum albumin (BSA), which has binding sites for fatty acids and may also serve as a cholesterol shuttle in serum (Sankaranarayanan et al., 2013; van der Vusse, 2009). We hypothesized that fatty acids carried by albumin may become toxic following exposure to oxidative stress and low pH and that import of fatty acids into the bacteria might be a protective mechanism. . We therefore tested whether fatty acids are required for the susceptibility of Δoperon mutants using exposure to acid and oxidative stress in Sauton’s broth, a minimal medium that lacks BSA, and found that in the absence of fatty acids the Δoperon mutants persisted to the same degree as wild-type bacteria (Figure 3C). To test whether lipids bound to BSA mediate the susceptibility of DyP mutants to acid and oxidative stress, we cultured wild-type and Δoperon Mtb in media constituted with fatty acid-free BSA and exposed the bacteria to H₂O₂ at pH 4.5 for 3 days. In the absence of BSA-bound lipids, we found that the Δoperon mutant was not susceptible to oxidative stress at low pH (Figure 3D). 7H9 medium prepared with lipid-free BSA was then reconstituted with oleic acid, an abundant lipid found in mammalian systems (Abdelmagid et al., 2015). Reconstitution of fatty acid-free medium with oleic acid restored toxicity to nanocompartment mutants under conditions of acid and oxidative stress (Figure 3D). Taken together, these data suggest that the phagosomal lipids used by Mtb as a carbon source may be toxic to bacteria lacking functional nanocompartments under conditions of oxidative stress.

We considered the possibility that lipids present in the medium might disrupt Mtb membranes, resulting in altered pH homeostasis and a drop in cytosolic pH. Interestingly, in acidified medium we found that the intracellular pH of wild-type bacteria dropped from ~pH 7.5 to ~pH 6.2, and this decrease was dependent on the presence of albumin-bound fatty acids (Figure 3E). These data confirm published results demonstrating that the cytosolic pH of Mtb drops significantly during acid exposure (Vandal et al., 2008). A lower cytosolic pH would possibly impair the function of many other bacterial enzymes important for resisting oxidative stress and place increasing importance on enzymes that can function in acidic environments, such as DyP (Uchida et al., 2015). Taken together, these data may explain why functional nanocompartments are critical for bacterial survival when Mtb is exposed to acid and oxidative stress in a fatty acid-rich environment.

Our in vitro findings that DyP nanocompartments are important for defense against a combination of oxidative stress, low pH, and fatty acids suggested that this system may be important for Mtb survival in the phagosomal environment. Therefore, we sought to determine whether DyP nanocompartments are required for bacterial growth in host cells. We found that the Δoperon strain was consistently attenuated for growth in murine bone marrow-derived macrophages (Figure 4A). We attempted to complement this phenotype; however, the limited dynamic range between wild-type and mutant in macrophages made clean interpretation of the complementation results difficult. As these genes are predicted to be in a two-gene operon with no other predicted genes in close proximity, polar effects are extremely unlikely. However, to confirm that the phenotype of the mutants in macrophages is due to the loss of the two-gene encapsulin operon, we repeated the experiment with the independently derived Dyp:Tn mutant and also observed attenuated growth in macrophages across repeated experiments (Figure 4B).

Figure 4

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One of the antibiotics used to treat TB infection, PZA, is most effective in low pH environments, such as the phagolysosome. PZA is thought to disrupt the cell wall of Mtb, which leads to acidification of the bacterial cytosol. A recent study in human TB patients demonstrated that the pH of necrotic lung cavities is ~5.5, a finding that may also explain the efficacy of PZA in treating Mtb infection (Kempker et al., 2017). Since DyP is critical for protection of Mtb against oxidative stress at low pH, we considered whether nanocompartments may mediate Mtb resistance to PZA. To test bacterial susceptibility to PZA, we treated wild-type and Δoperon Mtb with PZA in the presence of H₂O₂ at pH 5.5. We found that mutants lacking the DyP nanocompartment were sensitive to concentrations of PZA that did not impact growth of the wild-type bacteria (Figure 4C). We next set out to test whether mutants lacking the DyP operon are susceptible to PZA treatment in vivo. We infected BALB/C mice with wild-type and mutant bacteria by the aerosol route with a dose of ~250 bacteria instilled on day 0 of infection. PZA treatment began on day 14 and continued until day 35, at which time we enumerated CFU in the lungs. We did not find that Δoperon mutants were more attenuated than wild-type bacteria in mouse lungs at 35 days post infection, at least under the conditions examined. However, Δoperon mutants were significantly more susceptible to treatment with PZA in vivo (Figure 4D), demonstrating that the DyP encapsulin operon is required for endogenous resistance to PZA treatment.

Here, we report that a protein nanocompartment in Mtb encapsulates the dye-decolorizing peroxidase DyP. By demonstrating that the encapsulin system is important for resisting oxidative stress at low pH, we provide the first evidence that encapsulin systems contribute to oxidative stress in a bacterial pathogen, and only the second demonstration of a functional encapsulin system in any species. Furthermore, we expand our understanding of the importance of encapsulation in enzyme function by demonstrating that the protein shell encapsulating DyP enhances its function in vivo. Finally, we demonstrate that the DyP nanocompartment system is essential for the ability of Mtb to grow in host macrophages and to resist killing by the antibiotic PZA. These findings are the first demonstration that a nanocompartment system is important for virulence in any bacterial pathogen.

Bacterial protein-based compartments are emerging as functional equivalents of eukaryotic organelles that can sequester enzymatic reactions from the environment of the cytosol. Bacteria appear to have two predominant classes of protein based organelles, the relatively well-understood bacterial microcompartment (Kerfeld et al., 2018) and the more recently discovered bacterial nanocompartment (Nichols et al., 2017). Rather than using a lipid membrane for compartmentalization, both bacterial microcompartments and nanocompartments consist of a porous protein shell that surrounds enzymatic proteins. Bacterial microcompartments that enable utilization of specialized nutrients including ethanolamine and 1,2 propane diol have previously been implicated in pathogenesis of E. coli and Salmonella enterica, respectively (Jakobson and Tullman-Ercek, 2016). Nanocompartments, which are 10 times smaller than a typical bacterial microcompartment, have been identified bioinformatically in over 900 bacterial species; however, very few studies have addressed the role that nanocompartments play in bacterial physiology. Bioinformatic analysis and the heterologous expression of nanocompartment systems from diverse bacteria in E. coli have suggested that these systems might function in iron mineralization, oxidative and nitrosative stress resistance, and anaerobic ammonium oxidation (Giessen and Silver, 2017). However, the only nanocompartment that has been shown to be important for survival under conditions of stress is the ferritin-like protein containing compartment in M. xanthus, which is required for resistance to H₂O₂ (McHugh et al., 2014). Our demonstration that an encapsulin system is required for resistance to oxidative stress in Mtb significantly broadens the potential role and impact of nanocompartment systems in bacterial physiology.

A previous report examined the Mtb nanocompartment by heterologous expression of the encapsulin gene in E. coli. Coexpression with three potential substrates identified bioinformatically based on a putative signal sequence suggested that three proteins can be encapsulated by the Mtb nanocompartment: DyP, FolB, and BrfB (Contreras et al., 2014). Here, we show for the first time that Mtb produces this nanocompartment. However, of the three potential cargo proteins, only DyP was identified in encapsulin systems purified from logarithmically growing bacteria. Why is the DyP protein encapsulated? There is almost no understanding of the role of encapsulation in bacterial physiology despite the widespread presence of encapsulins across bacterial species. In this study, we found that DypB alone can protect the bacteria from oxidative stress at low pH. It is possible that in some circumstances, particularly when the stressor is not maximal, encapsulation of DyP may not be necessary. We expect that further investigation of this nanocompartment system in Mtb and in other bacterial species will reveal additional roles of encapsulins in the bacterial physiology.

We showed that Mtb mutants lacking the DyP encapsulin system have an altered redox balance at baseline, with a significantly more oxidative cytosol than wild-type. However, under standard growth conditions, the mutants are not defective. While mutants exhibit a small defect for survival when exposed to H₂O₂ at pH 6.5, they are highly attenuated at low pH in the presence of H₂O₂. Interestingly, the attenuation of the mutants under these conditions requires the presence of fatty acids bound to BSA in the culture medium. Why is the DyP encapsulin system required for growth under these conditions? It is possible that this system is required for detoxifying an oxidative species other than H₂O₂ itself, such as a lipid peroxide. It is also possible that the low pH compromises the integrity of the cell wall such that oxidative damage is sustained, or that the low pH magnifies the importance of DyP, which has a low pH optimum. Nevertheless, although the exact function of the DyP encapsulin system remains to be elucidated, this system is clearly important for pathogenesis as mutants fail to grow in host macrophages. Intriguingly, encapsulin systems have been shown to confer stability to the enzymatic cargo under harsh conditions, including heat and protease exposure (Nichols et al., 2017). Cfp29 was originally identified as a component of short-term culture filtrate present in broth of growing cells, leading to the supposition that this protein is secreted (Rosenkrands et al., 1998). Given that nanocompartments spontaneously assemble inside cells (Cassidy-Amstutz et al., 2016; Nichols et al., 2017; Snijder et al., 2016), it is unlikely that a structure the size of a ribosome could be secreted. We speculate that nanocompartments are released from dying cells, and that their extreme stability promotes their accumulation in axenic bacterial culture. Indeed, this stability may partially explain why Cfp29 is a known immunodominant antigen for T cells (Rosenkrands et al., 1998). Taken together, these data suggest the intriguing possibility that nanocompartments released from dying cells in vivo could continue to provide defense against oxidative stress for the surviving bacteria.

We found that only two peroxidases, KatG and DyP, are required for survival in the presence of H₂O₂ at pH 4.5. These findings raise the possibility that these peroxidases have redundant function in vivo, possibly explaining the lack of attenuation we observed in Δoperon mutants in BALB/C mice at 35 days post infection. It is also possible that Δoperon mutants are not attenuated at this timepoint in BALB/C mice as the Tn-seq screen that previously identified Cfp29 as being required for growth in mouse lungs used the C57BL/6 strain (Zhang et al., 2013). KatG mutants are highly prevalent in the clinic as loss of function in KatG prevents activation of the INH prodrug. Importantly, many KatG mutants that fail to activate INH are also defective for catalase activity (DeVito and Morris, 2003; Mo et al., 2004; Wei et al., 2003). This raises the possibility that the DyP nanocompartment system could partially compensate for a loss of KatG catalase activity, enabling these drug resistant mutants to survive in vivo. In addition to a potential role for DyP in the context of INH treatment and INH resistant infections, we found that DyP encapsulin mutants are susceptible to treatment with PZA, an antibiotic known to require acidic conditions for efficacy. Thus, we predict that this encapsulin system may limit the efficacy of PZA treatment of patients.

In summary, we have demonstrated that Mtb produces a functional nanocompartment containing the peroxidase DyP. This system is essential for resisting oxidative stress at low pH, conditions that resemble the host lysosome. In addition, the DyP encapsulin system is required for growth in host macrophages and for resistance to the antibiotic PZA. Previously encapsulin systems have been primarily studied in the context of heterologous expression in E. coli and for bioengineering or structural studies. Our findings demonstrate the significance of encapsulin nanocompartments in bacterial physiology in the globally significant pathogen Mtb.

All data generated or analysed during this study are included in the manuscript and supporting file.

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[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "A nanocompartment containing the peroxidase DypB contributes to defense against oxidative stress in M. tuberculosis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Neeraj Dhar (Reviewer #2).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

As you will see from the detailed reviewer comments, there are several interesting aspects of the work but at this stage, there are notable limitations also. Given this, we are rejecting the manuscript in its current form. That said, your findings are compelling indeed and if you are able to address the concerns, I would be willing to reconsider the study as a new manuscript, where you indicate how you have dealt with the reviewer's comments from the first round of review.

Reviewer #1:

Lien and colleagues report on the role of encapsulin Cfp29/Rv0798c and peroxidase DypB/Rv0799c in the resistance of M. tuberculosis to oxidative stress. The study builds on a previous study (Contreras et al. 2014) which reported the detection of Cfp29-encapsulated DypB in recombinant E. coli; here, the authors show that encapsulation also occurs in M. tuberculosis. In addition, the authors show that a cfp29/dypB-null mutant is highly sensitive to H202 at acidic pH, and their data suggest that encapsulation of DypB increases peroxidase activity. Finally, the authors report that dypB- and cfp29/dypB-null mutants are altered in their ability to multiply within macrophages.

Overall, the concept of compartmentalization in prokaryotes is of great interest; perhaps the most interesting conclusion is that the encapsulation of a virulence factor contributes to resistance to host-imposed stress in a major pathogen, although the role of Cfp29 in enhancing DypB activity seems rather modest, both in vitro and in vivo. However, the study as a whole contains many results that are poorly related (see comments below), the reader is finally left with an attractive hypothesis that is not sufficiently explored mechanistically, and the study appears a bit preliminary in this respect. How are nanocapsules secreted? Are they present in the mycobacterial vacuole? Is the phenotype of dypB- and cfp29/dypB-null mutants in macrophages really related to oxidative stress? What is the phenotype of these mutants in vivo? Addressing these questions, and perhaps others, would have allowed a more comprehensive study, instead of including unrelated or poorly related data on KatG, lipids, etc.

– Data suggesting that "DypB encapsulation enhances protection against oxidative stress under low pH conditions" are not entirely convincing. In Figure 2D, the expression of DypB in the DypB::Tn mutant almost fully restores resistance to H₂O₂. Is the difference between DypB::Tn+pDypB and DypB::Tn+pOperon significant? Similarly, in Figure 2E, the phenotype of the cfp29-null mutant should be restored by genetic complementation.

– The phenotype of the Rv1762c mutant is quite clear (Figure 2F), but the mutant has not been complemented, and more importantly, the question of whether this is related to encapsulation is not explored at all; I suggest deleting this data as it adds nothing to the overall message.

– "Based on the Tn-seq data, we hypothesized that lipids may be responsible for the sensitivity of DypB mutants to oxidative stress under acidic conditions". I do not understand this hypothesis. The Tn-seq data reveal mutants that are attenuated under conditions of acidic and oxidative stress. As far as I understand, the experiment was not designed to identify the genes involved in the sensitivity of the DypB mutant, which would have required a library of Tn mutants in a dypB-null background.

– The effect of lipids on the phenotype of the operon-null mutant is clear; but the reader is given no further explanation here. The same applies for katG/INH; these results are poorly related to the study as a whole.

– The phenotype of both mutants in macrophages is quite modest and should be verified by genetic complementation.

Reviewer #2:

In this manuscript the authors describe and characterize the role of a two-gene operon predicted to encode for an encapsulated nanocompartment in M. tuberculosis. The authors show that heterologous expression of these genes in E. coli produces Cfp29 nanocompartments that encapsulate the DypB peroxidase and protect it from degradation. They also provide first evidence that Cfp29-DypB nanocompartments are produced by Mtb under normal growth conditions. Further characterization revealed that DypB had maximal activity at low pH, an environment that M. tuberculosis faces during its intracellular lifestyle. To determine if the encapsulated DypB serves a protective role against oxidative stress, M. tb strains carrying deletions of the DypB operon were tested against oxidative stress under normal and acidic conditions. The mutant strain was found to be more sensitive to oxidative stress especially in acidic environments. A transposon sequencing screening identified DypB and KatG as the only peroxidades required for survival to oxidative stress at low pH, together with other genes involved in lipid or cholesterol metabolism. Interestingly, the hypersusceptibility of the DypB mutant was found to be dependent on free lipids present in the culture medium. Use of a redox potential fluorescent reporter revealed the mutant to have a more oxidizing intracellular environment compared to wild-type bacteria. These observations leaded to the conclusion that DypB nanocompartments protect bacteria from oxidative stress, low pH, and fatty acids suggesting that this system may be important for survival in phagosomes. This hypothesis is proved by infecting bone-marrow derived macrophages with the WT and the double mutant strains and showing that the mutant is attenuated. Finally, the double mutant was also shown to be more susceptible to Pyrazinamide suggesting that nanocompartments are important for resistance to oxidative stress.

The manuscript is written well and the experiments have been planned and executed well, to demonstrate the role of the DypB peroxidase against oxidative stress.

The only major concern is the relatively lesser focus on the encapsulation aspect. The data showing the presence of nanocompartments in Mtb and the encapsulation of DypB is quite convincing. However, all the subsequent characterization and most of the follow-up experiments are done with the mutant strain lacking both the DypB as well as the cfp29 genes. The authors already have a strain (∆cfp29) that expresses an unencapsulated DypB ( or even DypB::tn+pDypB). These mutants should be included in the stress-susceptibility experiments shown in Figures 3 and 4. This set of experiments along with the ones with the deletion of the complete operon will allow the clearer understanding of the role of DypB and the process of encapsulation. Since the main focus of the manuscript is about nanocompartments, disentangling this is crucial to the message.

Reviewer #3:

In Lien et al., the authors investigate a nanocompartment in the human pathogen Mycobacterium tuberculosis and explore how the cargo DypB, a peroxidase, contributes to bacterial defense against oxidative and acidic stress. Towards this, the authors have isolated nanocompartments and identified DypB as cargo. They show that mutants lacking the nanocompartment are attenuated in vitro by H₂O₂ in an acidic environment in the presence of lipids. The authors propose an attractive hypothesis-- that the bacteria encapsulate DypB to prevent its degradation by host cellular proteases and that it plays a role in resisting oxidative and acidic stress that the bacteria encounter inside macrophages. They show that DypB and nanocompartment mutants are attenuated in their survival inside the macrophages, although they do not show that this is due to their enhanced susceptibility to pH and oxidative stress. Overall, this is an interesting manuscript which has the potential to add important insight into encapsulation systems and the defense mechanisms that M. tuberculosis employs to survive inside acid and ROS rich phagosomes. The work, however, seems preliminary. It could be substantially strengthened by additional macrophage experiments, which currently lack important controls and are under-developed, as well as in vivo experiments.

1. The macrophage experiments need to include the complemented strains.

2. The macrophage experiments should include conditions that alter ROS, such as IFN-γ treatment, Nox2 knockouts, ROS neutralizing agents. It would also be informative to include pH and/or cathepsin neutralizing treatments. The Mrrx-GFP reporter could also be used to assess the redox state of the bacteria in macrophages.

3. The authors should determine whether the encapsulation mutant is attenuated in macrophages.

4. What contribution does DypB and the encapsulation system make to virulence in vivo?

5. It remains unclear how nanocompartments get to the extracellular space or if that is where they function. It would be useful to determine whether purified nanocompartments can rescue the mutant in vitro and in macrophages.

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

Thank you for submitting your article "A nanocompartment system contributes to defense against oxidative stress in M. tuberculosis" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Bavesh Kana 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:

1. Statistics should be provided in Figure 3E.

2. There seems to be a mis-annotation in Figure 4B.

3. Complementation in Figure 4A,B was difficult and the authors should clearly state their arguments in the manuscript (as they did in the reply to reviewers comment), so as not to leave a gap in this regard.

4. Legend to Figure 2: Initially it is mentioned that in panels C, E and F, the bacteria were exposed to 2. 5 mM H₂O₂ for 72 hours. Later it is mentioned that in D, E, F they were exposed for 24 hours. In the text it is mentioned that the bacteria were exposed for 72h. Clarify which one is accurate. Also, panel F is listed as representative of 2 or 3 experiments. Which one is correct? In panel 2C, the fourth bar is labelled as DypB. Should be DyP. For consistency, please plot y-axis in 2F in log scale.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

Lien and colleagues report on the role of encapsulin Cfp29/Rv0798c and peroxidase DypB/Rv0799c in the resistance of M. tuberculosis to oxidative stress. The study builds on a previous study (Contreras et al. 2014) which reported the detection of Cfp29-encapsulated DypB in recombinant E. coli; here, the authors show that encapsulation also occurs in M. tuberculosis. In addition, the authors show that a cfp29/dypB-null mutant is highly sensitive to H202 at acidic pH, and their data suggest that encapsulation of DypB increases peroxidase activity. Finally, the authors report that dypB- and cfp29/dypB-null mutants are altered in their ability to multiply within macrophages.

Overall, the concept of compartmentalization in prokaryotes is of great interest; perhaps the most interesting conclusion is that the encapsulation of a virulence factor contributes to resistance to host-imposed stress in a major pathogen, although the role of Cfp29 in enhancing DypB activity seems rather modest, both in vitro and in vivo. However, the study as a whole contains many results that are poorly related (see comments below), the reader is finally left with an attractive hypothesis that is not sufficiently explored mechanistically, and the study appears a bit preliminary in this respect. How are nanocapsules secreted? Are they present in the mycobacterial vacuole? Is the phenotype of dypB- and cfp29/dypB-null mutants in macrophages really related to oxidative stress? What is the phenotype of these mutants in vivo? Addressing these questions, and perhaps others, would have allowed a more comprehensive study, instead of including unrelated or poorly related data on KatG, lipids, etc.

– Data suggesting that "DypB encapsulation enhances protection against oxidative stress under low pH conditions" are not entirely convincing. In Figure 2D, the expression of DypB in the DypB::Tn mutant almost fully restores resistance to H₂O₂. Is the difference between DypB::Tn+pDypB and DypB::Tn+pOperon significant? Similarly, in Figure 2E, the phenotype of the cfp29-null mutant should be restored by genetic complementation.

The reviewer correctly points out that DypB accounts for the majority of the phenotype when TB is exposed to H₂O₂ at low pH. However, we do observe that Cfp29 also makes a smaller, yet reproducible and statistically significant contribution. To make this more convincing we have added the significance analysis to figure 2D. We have also complemented the Cfp29 mutant as requested, new Figure 2F. Given that there is a lack of any demonstration in the literature that an encapsulin protein is important in the physiology of a bacterium, despite the widespread presence of encapsulins in hundreds of bacterial species, we believe that this result, though somewhat modest, is important.

– The phenotype of the Rv1762c mutant is quite clear (Figure 2F), but the mutant has not been complemented, and more importantly, the question of whether this is related to encapsulation is not explored at all; I suggest deleting this data as it adds nothing to the overall message.

We thank the reviewer for pointing this out and have removed the data as suggested.

– "Based on the Tn-seq data, we hypothesized that lipids may be responsible for the sensitivity of DypB mutants to oxidative stress under acidic conditions". I do not understand this hypothesis. The Tn-seq data reveal mutants that are attenuated under conditions of acidic and oxidative stress. As far as I understand, the experiment was not designed to identify the genes involved in the sensitivity of the DypB mutant, which would have required a library of Tn mutants in a dypB-null background.

The reviewer is correct that the Tn-seq experiment was conducted to identify peroxidases required under conditions of oxidative stress at low pH. However, because Tn-Seq is comprehensive, we also identified a wider set of genes required for resistance under these conditions. We were intrigued by our observation that mutants encoding multiple subunits of the Mce1 fatty acid importer were susceptible under these conditions. This led us to hypothesize that importing fatty acids might protect the bacteria from fatty acid toxicity generated under oxidative stress at low pH. This caused us to test whether eliminating fatty acids from the medium entirely eliminated the effect, which we did observe. We realize we did not explain this rationale clearly in the text, so we thank the reviewer for raising this question. We have revised the text to be more clear.

– The effect of lipids on the phenotype of the operon-null mutant is clear; but the reader is given no further explanation here. The same applies for katG/INH; these results are poorly related to the study as a whole.

Again, we thank the reviewer for pointing out insufficient explanation of the results we have in the text – we have added more discussion and context. However, we do acknowledge that we do not yet understand the role of fatty acids in the phenotype we describe. This is a recently discovered biological system about which almost nothing is known in any bacterial species, and there is much we need to learn moving forward. However, we would argue that it is beyond the scope of the first publication on the endogenous system in Mtb to fully elucidate the mechanism.

– The phenotype of both mutants in macrophages is quite modest and should be verified by genetic complementation.

The reviewer correctly points out that the phenotype is modest in macrophages. The phenotypes of TB bacterial mutants are often modest in macrophages, given the limited dynamic range of growth. We have attempted to complement these results however, complementation of the nanocompartment system has proven to be difficult, particularly in macrophages. The limited dynamic range between wild-type and mutant in macrophages also makes interpretation of the complementation results tricky.

To confirm that the phenotype of these mutants in macrophages is due to the loss of the two gene encapsuling operon, we performed this experiment with two independently derived mutants >5 times, and always observe attenuation of both mutants in macrophages.

Furthermore, these genes are predicted to be in a 2 gene operon with no other predicted genes in close proximity, therefore polar effects are extremely unlikely. We therefore stand by this data and would like to include it in the final manuscript, even though we have not observed attenuation in vivo. We believe that this is important data for other researchers in the field who may be interested in examining nanocompartments in other bacterial pathogens.

Reviewer #2:

In this manuscript the authors describe and characterize the role of a two-gene operon predicted to encode for an encapsulated nanocompartment in M. tuberculosis. The authors show that heterologous expression of these genes in E. coli produces Cfp29 nanocompartments that encapsulate the DypB peroxidase and protect it from degradation. They also provide first evidence that Cfp29-DypB nanocompartments are produced by Mtb under normal growth conditions. Further characterization revealed that DypB had maximal activity at low pH, an environment that M. tuberculosis faces during its intracellular lifestyle. To determine if the encapsulated DypB serves a protective role against oxidative stress, M. tb strains carrying deletions of the DypB operon were tested against oxidative stress under normal and acidic conditions. The mutant strain was found to be more sensitive to oxidative stress especially in acidic environments. A transposon sequencing screening identified DypB and KatG as the only peroxidades required for survival to oxidative stress at low pH, together with other genes involved in lipid or cholesterol metabolism. Interestingly, the hypersusceptibility of the DypB mutant was found to be dependent on free lipids present in the culture medium. Use of a redox potential fluorescent reporter revealed the mutant to have a more oxidizing intracellular environment compared to wild-type bacteria. These observations leaded to the conclusion that DypB nanocompartments protect bacteria from oxidative stress, low pH, and fatty acids suggesting that this system may be important for survival in phagosomes. This hypothesis is proved by infecting bone-marrow derived macrophages with the WT and the double mutant strains and showing that the mutant is attenuated. Finally, the double mutant was also shown to be more susceptible to Pyrazinamide suggesting that nanocompartments are important for resistance to oxidative stress.

The manuscript is written well and the experiments have been planned and executed well, to demonstrate the role of the DypB peroxidase against oxidative stress.

The only major concern is the relatively lesser focus on the encapsulation aspect. The data showing the presence of nanocompartments in Mtb and the encapsulation of DypB is quite convincing. However, all the subsequent characterization and most of the follow-up experiments are done with the mutant strain lacking both the DypB as well as the cfp29 genes. The authors already have a strain (∆cfp29) that expresses an unencapsulated DypB ( or even DypB::tn+pDypB). These mutants should be included in the stress-susceptibility experiments shown in Figures 3 and 4. This set of experiments along with the ones with the deletion of the complete operon will allow the clearer understanding of the role of DypB and the process of encapsulation. Since the main focus of the manuscript is about nanocompartments, disentangling this is crucial to the message.

We agree with this point and have performed additional experiments with the Cfp29 mutant. First, we have shown that although the DyP mutant has defects in intracellular redox state at baseline, which is exacerbated under conditions of oxidative stress, we do not observe a change in redox state in a Cfp29 mutant (Figure 2G). We have added this to the text. In addition, we have not observed a phenotype of the Cfp29 mutant in exposure to PZA, however it is possible that is because we have not identified conditions under which the bacteria are most susceptible to this antibiotic. We do find consistently that the Cfp29 protein plays a small but significant role in protecting against exposure to oxidative stress at low pH, and we do believe that as we continue to explore this system we will better understand under which encapsulation is important for physiological function. It is important to note that we provide the first evidence that encapsulin systems contribute to oxidative stress in a bacterial pathogen, and only the second demonstration of a functional encapsulin system in any species. We therefore think these findings while exciting and informative for microbiologists across disciplines, are still by necessity preliminary due to the nascent status of the field.

We have expanded our discussion of the significance of these results in the Discussion section.

Reviewer #3:

In Lien et al., the authors investigate a nanocompartment in the human pathogen Mycobacterium tuberculosis and explore how the cargo DypB, a peroxidase, contributes to bacterial defense against oxidative and acidic stress. Towards this, the authors have isolated nanocompartments and identified DypB as cargo. They show that mutants lacking the nanocompartment are attenuated in vitro by H₂O₂ in an acidic environment in the presence of lipids. The authors propose an attractive hypothesis-- that the bacteria encapsulate DypB to prevent its degradation by host cellular proteases and that it plays a role in resisting oxidative and acidic stress that the bacteria encounter inside macrophages. They show that DypB and nanocompartment mutants are attenuated in their survival inside the macrophages, although they do not show that this is due to their enhanced susceptibility to pH and oxidative stress. Overall, this is an interesting manuscript which has the potential to add important insight into encapsulation systems and the defense mechanisms that M. tuberculosis employs to survive inside acid and ROS rich phagosomes. The work, however, seems preliminary. It could be substantially strengthened by additional macrophage experiments, which currently lack important controls and are under-developed, as well as in vivo experiments.

1. The macrophage experiments need to include the complemented strains.

Please see our comment to reviewer 1.

2. The macrophage experiments should include conditions that alter ROS, such as IFN-γ treatment, Nox2 knockouts, ROS neutralizing agents. It would also be informative to include pH and/or cathepsin neutralizing treatments. The Mrrx-GFP reporter could also be used to assess the redox state of the bacteria in macrophages.

We thank the reviewer for this suggestion. Although we have attempted numerous times to evaluate the phenotype of the mutants in phox deficient macrophages, we have observed inconsistent results. Although the mutant is always attenuated in wild-type macrophages, our results from the phox mutant are inconsistent. Similarly, we also attempted to measure the redox state of the bacteria in macrophages, but were unable to reproduce even the published data using this reporter in wild-type bacteria in macrophages, so have been unable to generate reliable data comparing wild-type and mutant bacteria.

3. The authors should determine whether the encapsulation mutant is attenuated in macrophages.

We have done these experiments, however due to the limited dynamic range of macrophage infections with Mtb we have inconsistent results. We have observed that the mutant is attenuated, but the differences are not statistically significant.

4. What contribution does DypB and the encapsulation system make to virulence in vivo?

We have performed an in vivo infection of BALB/C mice, due to the precedence for using this strain in studies of the antibiotic PZA, and do not see significant attenuation of the operon mutant. We are currently investigating whether attenuation of the mutant is mouse strain dependent (as Cfp29 was previously a hit in a transposon screen in C57BL/6 mice from the Rubin lab) and/or whether KatG can compensate for the loss of this system, however this will take some time to unravel and we feel is beyond the scope of the current manuscript.

5. It remains unclear how nanocompartments get to the extracellular space or if that is where they function. It would be useful to determine whether purified nanocompartments can rescue the mutant in vitro and in macrophages.

We have attempted these experiments and have seen a small effect of adding purified nanocompartments back in the in vitro system. However, we elected not to include this data as we feel it is difficult to interpret. First, it is difficult to purify sufficient material to achieve concentrations of nanocompartment that may be achieved within a phagosome. Second, we feel that it is difficult to extrapolate the biological significance of a positive result, as providing an enzyme that can function as a catalase should protect bacteria from oxidative stress, regardless of whether it may function naturally outside the cell. We do not feel that this is an important point of the story at this time, and for the sake of clarity prefer to omit the data.

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

Essential revisions:

1. Statistics should be provided in Figure 3E.

We have included statistics and thank the reviewers for pointing out this oversight.

2. There seems to be a mis-annotation in Figure 4B.

We have corrected the mis-annotated symbol.

3. Complementation in Figure 4A,B was difficult and the authors should clearly state their arguments in the manuscript (as they did in the reply to reviewers comment), so as not to leave a gap in this regard.

We have added an explanation to the text as requested.

4. Legend to Figure 2: Initially it is mentioned that in panels C, E and F, the bacteria were exposed to 2. 5 mM H₂O₂ for 72 hours. Later it is mentioned that in D, E, F they were exposed for 24 hours. In the text it is mentioned that the bacteria were exposed for 72h. Clarify which one is accurate. Also, panel F is listed as representative of 2 or 3 experiments. Which one is correct? In panel 2C, the fourth bar is labelled as DypB. Should be DyP. For consistency, please plot y-axis in 2F in log scale.

We apologize for the confusion and have clarified all of the requested information. The correct timepoint is 72h; 2F was done twice and 2G was done three times; we have changed DypB to Dyp; we have changed 2F to a log scale as requested.

Author details

1. Katie A Lien

   Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Kayla Dinshaw

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2721-0887

2. Kayla Dinshaw

   Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Katie A Lien

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5354-746X

3. Robert J Nichols

   Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8476-0554

4. Caleb Cassidy-Amstutz

   Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Matthew Knight

   Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Rahul Singh

   Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada

   Contribution

   Methodology, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

7. Lindsay D Eltis

   Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada

   Contribution

   Conceptualization, Methodology, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

8. David F Savage

   Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Investigation, Methodology, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0042-2257

9. Sarah A Stanley

   1. Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, United States
   2. School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   sastanley@berkeley.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4182-9048

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