A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C‐terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail‐forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane‐defined vacuoles. Functional complementation studies indicated that the C‐terminus, but not the N‐terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non‐pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.     

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome–lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C‐terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non‐pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.     

Rab10 Regulates Phagosome Maturation and Its Overexpression Rescues Mycobacterium‐Containing Phagosomes Maturation

Phagosome maturation follows a defined biochemical program and, in the vast majority of cases, the microbe inside the phagosome is killed and digested. Although, an important number of pathogens, including Mycobacterium tuberculosis, which kills around two million people every year, have acquired the ability to survive, and even replicate by arresting phagosomal maturation. To identify more of the machinery involved in phagocytosis and phagosomal maturation, we investigated the function of Rab10 in engulfment and maturation of inert particles and Mycobacterium bovis bacille Calmette‐Guérin (BCG). We showed that Rab10 association with phagosomes is transient and confocal microscopy revealed detectible levels of Rab10 on phagosomal membranes at very early time‐points, occurring even before Rab5 acquisition. Rab10 recruitment had strong functional consequence, as the knockdown of endogenous Rab10 by RNA interference or overexpression of Rab10 dominant‐negative mutant delayed maturation of phagosomes of IgG‐opsonized latex beads or heat killed‐mycobacteria. These results can be explained, at least in part, by the involvement of Rab10 in recycling of some phagosomal components. More importantly, overexpression of the constitutively active mutant of Rab10 partially rescued live‐Mycobacterium‐containing phagosomes maturation. Indeed, we found that the membrane harbouring Mycobacterium acquired early endosome antigen 1 (EEA‐1), a marker excluded from phagosomes in control cells. Altogether these results indicate that Rab10, acting upstream of Rab5, plays a prominent role in phagolysosome formation and can modulate Mycobacterium‐containing phagosomes maturation.     

Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant-negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb-containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.     

A lipoprotein modulates activity of the MtrAB two‐component system to provide intrinsic multidrug resistance,cytokinetic control and cell wall homeostasis in Mycobacterium

The MtrAB signal transduction system, which participates in multiple cellular processes related to growth and cell wall homeostasis, is the only two‐component system known to be essential in Mycobacterium. In a screen for antibiotic resistance determinants in Mycobacterium smegmatis, we identified a multidrug‐sensitive mutant with a transposon insertion in lpqB, the gene located immediately downstream of mtrA–mtrB. The lpqB mutant exhibited increased cell–cell aggregation and severe defects in surface motility and biofilm growth. lpqB cells displayed hyphal growth and polyploidism, reminiscent of the morphology of Streptomyces, a related group of filamentous Actinobacteria. Heterologous expression of M. tuberculosis LpqB restored wild‐type characteristics to the lpqB mutant. LpqB interacts with the extracellular domain of MtrB, and influences MtrA phosphorylation and promoter activity of dnaA, an MtrA‐regulated gene that affects cell division. Furthermore, in trans expression of the non‐phosphorylated, inactive form of MtrA in wild‐type M. smegmatis resulted in phenotypes similar to those of lpqB deletion, whereas expression of the constitutively active form of MtrA restored wild‐type characteristics to the lpqB mutant. These results support a model in which LpqB, MtrB and MtrA form a three‐component system that co‐ordinates cytokinetic and cell wall homeostatic processes.     

Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching

During phagosome maturation, the late endosomal marker Rab7 and the lysosomal marker LAMP1 localize to the phagosomes. We investigated the mobility of Rab7 and LAMP1 on the phagosomes in macrophages by fluorescence recovery after photobleaching (FRAP) analysis. Rab7 was mobile between the phagosomal membrane and the cytosol in macrophages that ingested latex beads during phagosome maturation. The addition of interferon-γ (IFN-γ) restricted this mobility, suggesting that Rab7 is forced to bind to the phagosomal membrane by IFN-γ-mediated activation. Immobilization of LAMP1 on the phagosomes was observed irrespective of IFN-γ-activation. We further examined the mobility of Rab7 on the phagosomes containing Mycobacterium bovis BCG by FRAP analysis. The rate of fluorescence recovery for Rab7 on mycobacterial phagosomes was lower than that on the phagosomes containing latex beads, suggesting that mycobacteria impaired the mobility of Rab7 and arrested phagosome maturation.     

Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in the context of innate immunity

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC‐MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.     

The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation

Phagocytosis by cells of the innate immune system, such as macrophages, and the subsequent successful maturation of the phagosome, is key for the clearance of pathogens. The fungal pathogen Cryptococcus neoformans is known to overcome killing by host phagocytes and both replicate within these cells and also escape via a non‐lytic process termed vomocytosis. Here we demonstrate that, during intracellular growth, cryptococci modify phagolysosome maturation. Live cryptococci, but not heat‐killed pathogens or inert targets, induce the premature removal of the early phagosome markers Rab5 and Rab11. In addition, significant acidification of the phagosome, calcium flux and protease activity is hindered, thus rendering the phagosome permissive for cryptococcal proliferation. Interestingly, several attenuated cryptococcal mutants retain this ability to subvert phagosomal maturation, suggesting that hitherto unidentified pathogen mechanisms regulate this process.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron

Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.     

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium‐containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid‐bound mycobactin (MBT) and the water‐soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe‐MBT or Fe‐cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe‐cMBT promoted the growth of wild‐type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild‐type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra‐ and intracellular growth of the pathogen.     

Drosophila Rab14 mediates phagocytosis in the immune response to Staphylococcus aureus

Drosophila haemocytes are essential for the animal to resist Staphylococcus aureus infections. Phagocytosis is a central component of the haemocyte‐mediated immune response. It involves regulated interaction between the phagocytic and the endocytic compartments. RabGTPases are pivotal for the membrane trafficking and fusion events, and thus are often targets of intracellular pathogens that subvert phagocytosis. An in vivo screen identified Rab2 and Rab14 as candidates for proteins regulating phagosome maturation. Since Rab14 is often targeted by intracellular pathogens, an understanding of its function during phagocytosis and the overall immune response can give insight into the pathogenesis of intracellular microbes. We generated a Drosophila Rab14 mutant and characterized the resulting immune defects in animals and specifically in haemocytes in response to an S. aureus infection. Haemocyte based immunofluorescence studies indicate that Rab14 is recruited to the phagosome and like Rab7, a well‐characterized regulator of the phagocytic pathway, is essential for progression of phagosome maturation. Rab14 mutant haemocytes show impaired recruitment of Rab7 and of a lysosomal marker onto S. aureus phagosomes. The defect in phagocytosis is associated with higher bacterial load and increased susceptibility to S. aureus in the animal.     

Dissection of Rab7 localization on Mycobacterium tuberculosis phagosome

The late endosomal marker Rab7 has been long believed to be absent from the phagosome containing Mycobacterium tuberculosis (M.tb) in macrophage, but the detail kinetics remains elusive. Here, we found that Rab7 is transiently recruited to and subsequently released from M.tb phagosomes. For further understanding of the effect of Rab7 dissociation from the phagosome, we examined the localization of lysosomal markers on the phagosome in the macrophage expressing a dominant-negative Rab7. The localization of lysosomal associated membrane protein-2 (LAMP-2) on the phagosome was Rab7-independent, while that of cathepsin D was Rab7-dependent. These results agree with the localization of each lysosomal marker on M.tb phagosome at 6 h postinfection-i.e., LAMP-2, but not cathepsin D localized on the majority of M.tb phagosomes. These results suggest that the dissociation of Rab7 from M.tb phagosome is the important process in inhibition of phagolysosome biogenesis.     

Proteomics approach to understand reduced clearance of mycobacteria and high viral titers during HIV–mycobacteria co‐infection

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV‐infected population. These seemingly harmless non‐pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV‐associated complications. Although immune‐evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome‐enriched fractions from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) mono‐infected and HIV–M. bovis BCG co‐infected THP‐1 cells by LC‐MALDI‐MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co‐infection helped the survival of non‐pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co‐infection up‐regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co‐infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co‐infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.     

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.     

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non‐pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGΔkef), deficient in a Kef‐type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild‐type BCG, and the production of ROS by macrophages infected with BCGΔkef is substantially decreased compared with those infected with wild‐type BCG. Our results suggest that increased intracellular survival of BCGΔkef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS.     

Porins facilitate nitric oxide-mediated killing of mycobacteria

Non-pathogenic mycobacteria such us Mycobacterium smegmatis reside in macrophages within phagosomes that fuse with late endocytic/lysosomal compartments. This sequential fusion process is required for the killing of non-pathogenic mycobacteria by macrophages. Porins are proteins that allow the influx of hydrophilic molecules across the mycobacterial outer membrane. Deletion of the porins MspA, MspC and MspD significantly increased survival of M. smegmatis in J774 macrophages. However, the mechanism underlying this observation is unknown. Internalization of wild-type M. smegmatis (SMR5) and the porin triple mutant (ML16) by macrophages was identical indicating that the viability of the porin mutant in vivo was enhanced. This was not due to effects on phagosome trafficking since fusion of phagosomes containing the mutant with late endocytic compartments was unaffected. Moreover, in ML16-infected macrophages, the generation of nitric oxide (NO) was similar to the wild type-infected cells. However, ML16 was significantly more resistant to the effects of NO in vitro compared to SMR5. Our data provide evidence that porins render mycobacteria vulnerable to killing by reactive nitrogen intermediates within phagosomes probably by facilitating uptake of NO across the mycobacterial outer membrane.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

Analysis of SecA2‐dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2‐dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2‐dependent substrates. Immunoblotting procedures confirmed SecA2‐dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG‐overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2‐dependent membrane localization of PknG is an important determinant for M. marinum virulence.     

The PGRS domain is responsible for translocation of PE_PGRS30 to cell poles while the PE and the C-terminal domains localize it to the cell wall

PE_PGRS proteins localize in the mycobacterial cell wall and the cell wall localization of PE_PGRS33 has been shown to be attributed to its PE domain. In this study, we expressed deletion mutants of PE_PGRS30 in Mycobacterium smegmatis to characterize the role of its domains in protein localization. It was revealed that, apart from the PE domain, the C-terminal domain present in few PE_PGRS proteins carries individual cell wall localization signals. Proteinase K sensitivity assay showed that PE_PGRS30 is exposed on the mycobacterial surface through its PGRS domain. PGRS domain was also shown to be responsible for polar localization of PE_PGRS30.