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Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in Bacillus subtilis are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates. Which cells express and deploy the various T7SS substrates is still unknown. To identify which cells express at least one of the T7SS substrates, we investigated the yfj operon. The yfjABCDEF operon encodes at least one predicted T7SS substrate. Starting with an in silico analysis of the yfj operon promoter region, we identified potential regulatory sequences. Using a yfj promoter-reporter fusion, we then identified several regulators that impact expression of the operon, including a regulator of biofilm formation, DegU. In a degU deletion mutant, yfj expression is completely abolished. Mutation of predicted DegU binding sites also results in a significant reduction in yfj reporter levels. Further analysis of yfj regulation reveals that deletion of spo0A has the opposite effect of the degU deletion. Following the yfj reporter by microscopy of cells harvested from biofilms, we find that the yfj operon is expressed specifically in the subset of cells undergoing sporulation. Together, our results define cells entering sporulation as the subpopulation most likely to express products of the yfj operon in B. subtilis .IMPORTANCEDifferential expression of genes in a bacterial community allows for the division of labor among cells in the community. The toxin substrates of the type VII secretions system (T7SS) are known to be active in Bacillus subtilis biofilm communities. This work describes the expression of one of the T7SS-associated operons, the yfj operon, which encodes the YFJ toxin, in the sporulating subpopulation within a biofilm. The evidence that the YFJ toxin may be deployed specifically in cells at the early stages of sporulation provides a potential role for deployment of T7SS in community-associated activities, such as cannibalism., Competing Interests: The authors declare no conflict of interest.

The VII secretion system is the main channel for Mycobacterium tuberculosis ( MTB ) to secrete virulence proteins. The ESAT-like proteins EsxA/B and EsxW/V in the RD region of its genome have been used as targets for vaccine antigens. However, the function of EsxO/P has not been explored, although it was predicted to potentially induce Th1 cell responses as a vaccine development target. In this study, the VII secretion system effector molecule Rv2347c was heterologously expressed in Mycobacterium smegmatis and found to inhibit the expression of the early marker RAB5 of phagosomes, thus preventing the maturation process of phagosomes toward lysosomes, and activated the host cytoplasmic sensing pathway. It inhibited autophagy and activated IFNβ transcription through the STING/TBK1 pathway promoting the host's survival. Therefore, Rv2347c plays an important role in the pathogenesis of MTB with the potential to be utilized as a new target for tuberculosis vaccine development., Importance: We found that the ESAT-like protein Rv2347c (EsxP) can inhibit the maturation of phagosomes, leading to mycobacterium escape from phagosomes into the cytoplasm, which triggers the host's cytoplasmic sensing pathway STING/TBK1, inhibiting autophagy and upregulating IFNβ transcription, which contributes to the survival of mycobacterium in the host cell. We also found that Rv2347c was able to activate host immunity by activating NF-κB via STING and promoting the transcription of downstream pro-inflammatory factors. Meanwhile, the host also produces IL-1β to repair phagosome maturation arrest via the STING-mediated non-NF-κB pathway., Competing Interests: The authors declare no conflict of interest.

Group B Streptococcus (GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia, and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has the potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Bacillota and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiont Enterococcus faecalis both in vitro using predator-prey assays and in vivo in the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named group B streptococcal L XG T oxin A ) contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of intracellular expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.IMPORTANCECompetition between neighboring, non-kin bacteria is essential for microbial niche establishment in mucosal environments. Gram-positive bacteria encoding T7SSb have been shown to engage in competition through the export of LXG-motif-containing toxins, but these have not been characterized in group B Streptococcus (GBS), an opportunistic colonizer of the polymicrobial female genital tract. Here, we show a role for GBS T7SS in competition with mucosal pathobiont Enterococcus faecalis , both in vitro and in vivo . We further find that a GBS LXG protein contributing to this antagonism is exported by the T7SS and is intracellularly toxic to other bacteria; therefore, we have named this protein group B streptococcal L XG T oxin A (BltA). Finally, we show that BltA and its associated chaperones promote persistence within female genital tract tissues, in vivo . These data reveal previously unrecognized mechanisms by which GBS may compete with other mucosal opportunistic pathogens to persist within the female genital tract., Competing Interests: The authors declare no conflict of interest.

Background: Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished., Methods: We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar., Results: Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567., Conclusion: B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA., (© 2024. The Author(s).)

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo . Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g , two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture., Importance: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo . The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection., Competing Interests: The authors declare no conflict of interest.

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.

Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

Staphylococcus aureus ( S. aureus ) can evade antibiotics and host immune defenses by persisting within infected cells. Here, we demonstrate that in infected host cells, S. aureus type VII secretion system (T7SS) extracellular protein B (EsxB) interacts with the stimulator of interferon genes (STING) protein and suppresses the inflammatory defense mechanism of macrophages during early infection. The binding of EsxB with STING disrupts the K48-linked ubiquitination of EsxB at lysine 33, thereby preventing EsxB degradation. Furthermore, EsxB-STING binding appears to interrupt the interaction of 2 vital regulatory proteins with STING: aspartate-histidine-histidine-cysteine domain-containing protein 3 (DHHC3) and TNF receptor-associated factor 6. This persistent dual suppression of STING interactions deregulates intracellular proinflammatory pathways in macrophages, inhibiting STING's palmitoylation at cysteine 91 and its K63-linked ubiquitination at lysine 83. These findings uncover an immune-evasion mechanism by S. aureus T7SS during intracellular macrophage infection, which has implications for developing effective immunomodulators to combat S. aureus infections., Competing Interests: Competing interests statement:The authors declare no competing interest.

Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum , the ESAT-6 system 1 (ESX-1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the espM or whiB6 gene does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by M. marinum . In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of whiB6 and ESX-1 genes during both laboratory growth and macrophage infection. EspN is also independently required for M. marinum growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis.IMPORTANCEPathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions., Competing Interests: The authors declare no conflict of interest.

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins., Competing Interests: Competing interests statement:The authors declare no competing interest.

Mycobacteria are microorganisms distributed in the environment worldwide, and some of them, such as Mycobacterium tuberculosis or M. leprae, are pathogenic. The hydrophobic mycobacterial cell envelope has low permeation and bacteria need to export products across their structure. Mycobacteria possess specialized protein secretion systems, such as the Early Secretory Antigenic Target 6 secretion (ESX) system. Five ESX loci have been described in M. tuberculosis, called ESX-1 to ESX-5. The ESX-3 secretion system has been associated with mycobacterial metabolism and growth. The locus of this system is highly conserved across mycobacterial species. Metallo-proteins regulate negative ESX-3 transcription in high conditions of iron and zinc. Moreover, this secretion system is part of an antioxidant regulatory pathway linked to Zinc. EccA3, EccB3, EccC3, EccD3, and EccE3 are components of the ESX-3 secretion machinery, whereas EsxG-EsxH, PE5-PPE4, and PE15-PPE20 are proteins secreted by this system. In addition, EspG3 and MycP3 are complementary proteins involved in transport and proteolysis respectively. This system is associated to mycobacterial virulence by releasing the bacteria from the phagosome and inhibiting endomembrane damage response. Furthermore, components of this system inhibit the host immune response by reducing the recognition of M. tuberculosis-infected cells. The components of the ESX-3 secretion system play a role in drug resistance and cell wall integrity. Moreover, the expression data of this system indicated that external and internal factors affect ESX-3 locus expression. This review provides an overview of new findings on the ESX-3 secretion system, its regulation, expression, and functions., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Mycobacterium infection can affect the host's immune function by secreting extracellular effector proteins. ESX (or type VII) system plays an important role in the secretion of effector proteins. ESX system is the protein export system in mycobacteria and many actinomycetes. However, how ESX system secretes and underlying mechanism of action remain unclear. In this review, we introduce the components, function, classification of ESX system and the process of substrates transfer to the peripheral space via this system, and discuss the roles of ESX system in antibiotics resistance, persistence, host-phage interaction, new drug targets. We hope to provide insights into the discovery of new drugs and vaccine antigens for tuberculosis.

Mycobacteria utilize type VII secretion systems (T7SSs) to secrete proteins across their highly hydrophobic and diderm cell envelope. Pathogenic mycobacteria have up to five different T7SSs, called ESX-1 to ESX-5, which are crucial for growth and virulence. Here, we use a functionally reconstituted ESX-5 system in the avirulent species Mycobacterium smegmatis that lacks ESX-5, to define the role of each esx-5 gene in system functionality. By creating an array of gene deletions and assessing protein levels of components and membrane complex assembly, we observed that only the five components of the inner membrane complex are required for its assembly. However, in addition to these five core components, active secretion also depends on both the Esx and PE/PPE substrates. Tagging the PPE substrates followed by subcellular fractionation, surface labeling and membrane extraction showed that these proteins localize to the mycobacterial outer membrane. This indicates that they could play a role in secretion across this enigmatic outer barrier. These results provide the first full overview of the role of each esx-5 gene in T7SS functionality. IMPORTANCE Pathogenic mycobacteria, such as the notorious Mycobacterium tuberculosis , are highly successful as pathogens, in part due to their specific and diderm cell envelope, with a mycolic acid-containing outer membrane. The architecture of this highly impermeable membrane is little understood and the proteins that populate it even less so. To transport proteins across their cell envelope, mycobacteria employ a specialized transport pathway called type VII secretion. While recent studies have elucidated the type VII secretion membrane channel that mediates transport across the inner membrane, the identity of the outer membrane channel remains a black box. Here, we show evidence that specific substrates of the type VII pathway could form these channels. Elucidating the pathway and mechanism of protein secretion through the mycobacterial outer membrane will allow its exploitation for the development of novel mycobacterial therapeutics., Competing Interests: The authors declare no conflict of interest.

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intraspecies diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low interspecies and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Furthermore, we observe subtype-specific effects of GBS T7SS on host colonization, as CJB111 subtype I but not CNCTC 10/84 subtype III T7SS promotes GBS vaginal colonization. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions., (© 2023 John Wiley & Sons Ltd.)

Introduction: GBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios., Methods: GBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS., Results: Most GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC., Conclusions: We demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schindler, Rahav, Nissan, Valenci, Ravins, Hanski, Ment, Tekes-Manova and Maor.)

The type VIIb protein secretion system (T7SSb) is found in Bacillota (firmicute) bacteria and has been shown to mediate interbacterial competition. EssC is a membrane-bound ATPase that is a critical component of the T7SSb and plays a key role in substrate recognition. Prior analysis of available genome sequences of the foodborne bacterial pathogen Listeria monocytogenes has shown that although the T7SSb was encoded as part of the core genome, EssC could be found as one of seven different sequence variants. While each sequence variant was associated with a specific suite of candidate substrate proteins encoded immediately downstream of essC , many LXG-domain proteins were encoded across multiple essC sequence variants. Here, we have extended this analysis using a diverse collection of 37 930 L . monocytogenes genomes. We have identified a rare eighth variant of EssC present in ten L. monocytogenes lineage III genomes. These genomes also encode a large toxin of the rearrangement hotspot (Rhs) repeat family adjacent to essC8 , along with a probable immunity protein and three small accessory proteins. We have further identified nine novel LXG-domain proteins, and four additional chromosomal hotspots across L. monocytogenes genomes where LXG proteins can be encoded. The eight L. monocytogenes EssC variants were also found in other Listeria species, with additional novel EssC types also identified. Across the genus, species frequently encoded multiple EssC types, indicating that T7SSb diversity is a primary feature of the genus Listeria .

Mycobacterium tuberculosis ( Mtb ) is transmitted through aerosols and primarily colonizes within the lung. The World Health Organization estimates that Mtb kills ~1.4 million people every year. A key aspect that makes Mtb such a successful pathogen is its ability to overcome iron limitation mounted by the host immune response. In our previous studies, we have shown that Mtb can utilize iron from heme, the largest source of iron in the human host, and that it uses two redundant heme utilization pathways. In this study, we show that the ESX-4 type VII secretion system (T7SS) is necessary for extracellular heme uptake into the Mtb cell through both heme utilization pathways. ESX-4 influences the secretion of the culture filtrate proteins Rv0125 and Rv1085c, which are also necessary for efficient heme utilization. We also discovered that deletion of the alternative sigma factor SigM significantly reduced Mtb heme utilization through both pathways and predict that SigM is a global positive regulator of core heme utilization genes of both pathways. Finally, we present the first direct evidence that some mycobacterial PPE (proline-proline-glutamate motif) proteins of the PPE protein family are pore-forming membrane proteins. Altogether, we identified core components of both Mtb Heme utilization pathways that were previously unknown and identified a novel channel-forming membrane protein of Mtb . IMPORTANCE M. tuberculosis ( Mtb ) is completely dependent on iron acquisition in the host to cause disease. The largest source of iron for Mtb in the human host is heme. Here, we show that the ancestral ESX-4 type VII secretion system is required for the efficient utilization of heme as a source of iron, which is an essential nutrient. This is another biological function identified for ESX-4 in Mtb , whose contribution to Mtb physiology is poorly understood. A most exciting finding is that some mycobacterial PPE (proline-proline-glutamate motif) proteins that have been implicated in the nutrient acquisition are membrane proteins that can form channels in a lipid bilayer. These observations have far-reaching implications because they support an emerging theme that PPE proteins can function as channel proteins in the outer mycomembrane for nutrient acquisition. Mtb has evolved a heme uptake system that is drastically different from all other known bacterial heme acquisition systems.

The ESX-5 secretion system is essential for the viability and virulence of slow-growing pathogenic mycobacterial species. In this study, we identified a 1,2,4-oxadiazole derivative as a putative effector of the ESX-5 secretion system. We confirmed that this 1,2,4-oxadiazole and several newly synthesized derivatives inhibited the ESX-5-dependent secretion of active lipase LipY by Mycobacterium marinum ( M. marinum ). Despite reduced lipase activity, we did not observe a defect in LipY secretion itself. Moreover, we found that several other ESX-5 substrates, especially the high molecular-weight PE_PGRS MMAR_5294, were even more abundantly secreted by M. marinum treated with several 1,2,4-oxadiazoles. Analysis of M. marinum grown in the presence of different oxadiazole derivatives revealed that the secretion of LipY and the induction of PE_PGRS secretion were, in fact, two independent phenotypes, as we were able to identify structural features in the compounds that specifically induced only one of these phenotypes. Whereas the three most potent 1,2,4-oxadiazoles displayed only a mild effect on the growth of M. marinum or M. tuberculosis in culture, these compounds significantly reduced bacterial burden in M. marinum -infected zebrafish models. In conclusion, we report a 1,2,4-oxadiazole scaffold that dysregulates ESX-5 protein secretion.

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection., Competing Interests: The authors have declared that no competing interests exist.

The type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified in Staphylococcus aureus , EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identical esaG and tsaI immunity genes are encoded in all S. aureus strains. To investigate the evolution of esaG repertoires, we analysed the sequences of the tandem esaG genes and their encoded proteins. We identified three blocks of high sequence similarity shared by all esaG genes and identified evidence of extensive recombination events between esaG paralogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss and expansion of esaG genes in S. aureus genomes and we identified evidence of such events during evolution of strains in clonal complex 8. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence similarity locate to the 5' and 3' end of tipC genes, and we found strong evidence for recombination between tipC paralogues encoded by Streptococcus mitis BCC08. By contrast, we found only a single homology block across tsaI genes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.

Streptococcus agalactiae causes sepsis and meningitis in neonates, presenting substantial clinical challenges. Type VII secretion system (T7SS), an important secretion system identified in Mycobacterium sp. and Gram-positive bacteria, was recently characterized in S. agalactiae and considered to contribute to its virulence and pathogenesis. In the present study, 128 complete genomic sequences of S. agalactiae were retrieved from GenBank to build a public dataset, and their sequences, capsular types, and clonal complexes were determined. Polymerase chain reaction (PCR) screening and genomic sequencing were conducted in an additional clinical dataset. STs and capsular types were determined using PCR. Eleven different types of T7SS were detected with similarities in gene order but differences in gene content. Strains with incomplete T7SS or lack of T7SS were also identified. Deletion, insertion, and segmentation of T7SS might be related to insertion sequences. The genetic environment of T7SS in S. agalactiae was also investigated and different patterns were identified downstream the T7SS, which were related to the diversity of T7SS putative effectors. The T7SS demonstrated possible sequence type (ST)-dependent diversity in both datasets. This work elucidated detailed genetic characteristics of T7SS and its genetic environment in S. agalactiae and further identified its possible ST-dependent diversity, which gave a clue of its mode of transmission. Further investigations are required to elucidate the underlying mechanisms and its functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhou, Xie, Xu and Sun.)

Mycobacterium tuberculosis ( Mtb ) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH , require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Bridging Centrality (BriCe) is a popular measure that combines the Betweenness centrality and Bridging coefficient metrics to characterize nodes acting as a bridge among clusters. However, there were no implementations of the BriCe plugin that can be readily used in the GEPHI software or any other software dedicated to graph-based studies. In this paper, we present the BriCe plugin for GEPHI. It is available as a third-party functionality from the native GEPHI interface as a handy plugin to add; hence, no additional download and installation process is necessary. The BriCe plugin for GEPHI is open-source, and one can access the code through the GEPHI GitHub repository. As a use case of the BriCe plugin, we analyzed the genome of Mycobacterium tuberculosis H37Rv to identify biological explanations on why some proteins were ranked with top BriCe values? For instance, we were able to formulate a new hypothesis combining the predicted sub cellular localization and high BriCe values concerning lipopolysaccharides (LPS) exportation. Our hypothesis provides a possible link among proteins of a glycosyltransferase group and the type VII Secretion System. The Bridging Centrality plugin for GEPHI is an easy to use tool for analyzing complex graphs and draw novel insights from graphical data.

The type VII protein secretion system (T7SS) of Staphylococcus aureus is encoded at the ess locus. T7 substrate recognition and protein transport are mediated by EssC, a membrane-bound multidomain ATPase. Four EssC sequence variants have been identified across S. aureus strains, each accompanied by a specific suite of substrate proteins. The ess genes are upregulated during persistent infection, and the secretion system contributes to virulence in disease models. It also plays a key role in intraspecies competition, secreting nuclease and membrane-depolarizing toxins that inhibit the growth of strains lacking neutralizing immunity proteins. A genomic survey indicates that the T7SS is widely conserved across staphylococci and is encoded in clusters that contain diverse arrays of toxin and immunity genes. The presence of genomic islands encoding multiple immunity proteins in species such as Staphylococcus warneri that lack the T7SS points to a major role for the secretion system in bacterial antagonism.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis , to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis . In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus ; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

Mycobacterium abscessus is an emerging pathogen that is often refractory to antibiotic control. Treatment is further complicated by considerable variation among clinical isolates in both their genetic constitution and their clinical manifestations. Here, we show that the prophage and plasmid mobilome is a likely contributor to this variation. Prophages and plasmids are common, abundant, and highly diverse, and code for large repertoires of genes influencing virulence, antibiotic susceptibility, and defense against viral infection. At least 85% of the strains we describe carry one or more prophages, representing at least 17 distinct and diverse sequence "clusters," integrated at 18 different attB locations. The prophages code for 19 distinct configurations of polymorphic toxin and toxin-immunity systems, each with WXG-100 motifs for export through type VII secretion systems. These are located adjacent to attachment junctions, are lysogenically expressed, and are implicated in promoting growth in infected host cells. Although the plethora of prophages and plasmids confounds the understanding of M. abscessus pathogenicity, they also provide an abundance of tools for M. abscessus engineering. IMPORTANCE Mycobacterium abscessus is an important emerging pathogen that is challenging to treat with current antibiotic regimens. There is substantial genomic variation in M. abscessus clinical isolates, but little is known about how this influences pathogenicity and in vivo growth. Much of the genomic variation is likely due to the large and varied mobilome, especially a large and diverse array of prophages and plasmids. The prophages are unrelated to previously characterized phages of mycobacteria and code for a diverse array of genes implicated in both viral defense and in vivo growth. Prophage-encoded polymorphic toxin proteins secreted via the type VII secretion system are common and highly varied and likely contribute to strain-specific pathogenesis., (Copyright © 2021 Dedrick et al.)

CpnT, a NAD + glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT. IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis , causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection., (Copyright © 2021 Izquierdo Lafuente et al.)

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus , is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC . Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

Protein EccE 1 is an essential component of the mycobacterial ESX-1 secretion system, which plays a crucial part in the process of virulence factors secretion, especially for pathogenic mycobacteria such as Mycobacterium tuberculosis. While EccE 1 was previously postulated to be the inner membrane pore-forming unit of a membrane complex through which substrates are transported, the structural properties of EccE 1 remains to be explored. In the present study, systematic Site-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) spectroscopic studies was carried out to reveal the secondary structure and transmembrane topology of the N-terminal Domain of EccE 1 protein (EccE 1 -NTD) from M. smegmatis in detergent micelles. EPR-based mobility and accessibility analysis of the R1 side chain for 64 residue positions of EccE 1 -NTD indicates that the transmembrane domain adopts two α-helices spanning Phe7-Cys30 and Leu36-Ile54. A tentative structural topology model of EccE 1 -NTD embedded in membrane is also suggested based on EPR spectroscopic data in this study, which will provide further insights into this protein and the ESX secretion systems of mycobacteria., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.

Bacteriophages (phages) are being considered as alternative therapeutics for the treatment of multidrug resistant bacterial infections. Considering phages have narrow host-ranges, it is generally accepted that therapeutic phages will have a marginal impact on non-target bacteria. We have discovered that lytic phage infection induces transcription of type VIIb secretion system (T7SS) genes in the pathobiont Enterococcus faecalis. Membrane damage during phage infection induces T7SS gene expression resulting in cell contact dependent antagonism of different Gram positive bystander bacteria. Deletion of essB, a T7SS structural component, abrogates phage-mediated killing of bystanders. A predicted immunity gene confers protection against T7SS mediated inhibition, and disruption of its upstream LXG toxin gene rescues growth of E. faecalis and Staphylococcus aureus bystanders. Phage induction of T7SS gene expression and bystander inhibition requires IreK, a serine/threonine kinase, and OG1RF_11099, a predicted GntR-family transcription factor. Additionally, sub-lethal doses of membrane targeting and DNA damaging antibiotics activated T7SS expression independent of phage infection, triggering T7SS antibacterial activity against bystander bacteria. Our findings highlight how phage infection and antibiotic exposure of a target bacterium can affect non-target bystander bacteria and implies that therapies beyond antibiotics, such as phage therapy, could impose collateral damage to polymicrobial communities., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis , the cause of human tuberculosis, and Mycobacterium marinum , a nontubercular pathogen with a broad host range, require the ESX-1 secretion system for virulence. The ESX-1 system secretes proteins which cause phagosomal lysis within the macrophage via an unknown mechanism. As reported elsewhere (R. E. Bosserman et al., Proc Natl Acad Sci U S A 114:E10772-E10781, 2017, https://doi.org/10.1073/pnas.1710167114), we recently discovered that the ESX-1 system regulates gene expression in M. marinum This finding was confirmed in M. tuberculosis in reports by C. Sala et al. (PLoS Pathog 14:e1007491, 2018, https://doi.org/10.1371/journal.ppat.1007491) and A. M. Abdallah et al. (PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). We further demonstrated that a feedback control mechanism connects protein secretion to WhiB6-dependent expression of the esx-1 genes via an unknown mechanism. Here, we connect protein secretion and gene expression by showing for the first time that specific ESX-1 substrates have dual functions inside and outside the mycobacterial cell. We demonstrate that the EspE and EspF substrates negatively control esx-1 gene expression in the M. marinum cytoplasm through the conserved WhiB6 transcription factor. We found that EspE and EspF are required for virulence and promote lytic activity independently of the major EsxA and EsxB substrates. We show that the dual functions of EspE and EspF are conserved in the orthologous proteins from M. tuberculosis Our findings support a role for EspE and EspF in virulence that is independent of the EsxA and EsxB substrates and demonstrate that ESX-1 substrates have a conserved role in regulating gene expression., (Copyright © 2020 American Society for Microbiology.)

Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract, and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin-resistant and -sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical, and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro but did not contribute to colonization in vivo Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5, and 8 days postinoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract., (Copyright © 2020 American Society for Microbiology.)

Bacteria have evolved intricate secretion machineries for the successful delivery of large molecules across their cell envelopes. Such specialized secretion systems allow a variety of bacteria to thrive in specific host environments. In mycobacteria, type VII secretion systems (T7SSs) are dedicated protein transport machineries that fulfill diverse and crucial roles, ranging from metabolite uptake to immune evasion and subversion to conjugation. Since the discovery of mycobacterial T7SSs about 15 y ago, genetic, structural, and functional studies have provided insight into the roles and functioning of these secretion machineries. Here, we focus on recent advances in the elucidation of the structure and mechanism of mycobacterial T7SSs in protein secretion. As many of these systems are essential for mycobacterial growth or virulence, they provide opportunities for the development of novel therapies to combat a number of relevant mycobacterial diseases.

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX-5 system is unable to fully complement secretion in an M. marinum esx-5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC 5 , possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creating M. marinum-M. tuberculosis EccC 5 chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC 5 . This region connects NBD1 and NBD2 of EccC 5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC 5 , showed a distinct secretion profile. These results indicate that linker 2 is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC 5 ., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis . The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB 3 , EccC 3 , and EccE 3 and two copies of the EccD 3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters., Competing Interests: NP, NC, RN, DT, KM, CS, OR No competing interests declared, (© 2019, Poweleit et al.)

The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°., (open access.)

Over the long course of evolution from a probable environmental reservoir, the pathogen that we know today as Mycobacterium tuberculosis has become fully capable of adapting to the life inside host cells by evading and modifying their responses to infection. Factors contributing to the success of this pathogen are numerous and thanks to a large body of work accumulated over the past decades, we are closer to understanding the remarkable complexity of tuberculosis pathogenesis. The unique type VII secretion systems and various complex lipids of the cell envelope have emerged as some of the most important and most studied factors in this regard. This review attempts to summarize recent findings on these and other virulence factors, while discussing their evolution in different closely related tuberculosis-causing bacteria as well, with the aim of exploring the processes which led M. tuberculosis to becoming one of the deadliest infections agents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The causative agent of human tuberculosis, Mycobacterium tuberculosis , has a complex lipid-rich diderm envelope, which acts as a major barrier protecting the bacterium against the hostile environment inside the host cells. For the transfer of diverse molecules across this complex cell envelope, M. tuberculosis has a series of general and specialized protein secretion systems, characterized by the SecA general secretion pathway, the twin-arginine translocation pathway, and five specific ESX type VII secretion systems. In this review, we focus on the latter systems, known as ESX-1 to ESX-5, which were first discovered almost 20 years ago during the in silico analysis of the genome sequence of M. tuberculosis H37Rv. Since then, these systems have been the subject of highly dynamic research due to their involvement in several key biological processes and host-pathogen interactions of the tubercle bacilli.

Background: Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens., Results: We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association., Conclusion: We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.

Gram-positive bacteria deploy the type VII secretion system (T7SS) to facilitate interactions between eukaryotic and prokaryotic cells. In recent work, we identified the TelC protein from Streptococcus intermedius as a T7SS-exported lipid II phosphatase that mediates interbacterial competition. TelC exerts toxicity in the inner wall zone of Gram-positive bacteria; however, intercellular intoxication of sister cells does not occur because they express the TipC immunity protein. In the present study, we sought to characterize the molecular basis of self-protection by TipC. Using sub-cellular localization and protease protection assays, we show that TipC is a membrane protein with an N-terminal transmembrane segment and a C-terminal TelC-inhibitory domain that protrudes into the inner wall zone. The 1.9-Å X-ray crystal structure of a non-protective TipC paralogue reveals that the soluble domain of TipC proteins adopts a crescent-shaped fold that is composed of three α-helices and a seven-stranded β-sheet. Subsequent homology-guided mutagenesis demonstrates that a concave surface formed by the predicted β-sheet of TipC is required for both its interaction with TelC and its TelC-inhibitory activity. S. intermedius cells lacking the tipC gene are susceptible to growth inhibition by TelC delivered between cells; however, we find that the growth of this strain is unaffected by endogenous or overexpressed TelC, although the toxin accumulates in culture supernatants. Together, these data indicate that the TelC-inhibitory activity of TipC is only required for intercellularly transferred TelC and that the T7SS apparatus transports TelC across the cell envelope in a single step, bypassing the cellular compartment in which it exerts toxicity en route., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mycobacterial pathogens have evolved a unique secretory apparatus called the Type VII secretion system (T7SS) which comprises of five gene clusters designated as ESX1, ESX2, ESX3, ESX4, and ESX5. Of these the ESX3 T7SS plays an important role in the regulatory uptake of iron from the environment, thereby enabling the bacteria to establish successful infection in the host. However, ESX3 secretion system is conserved among all the mycobacterial species including the fast-growing nonpathogenic species M. smegmatis. Although the function of ESX3 T7SS is known to be absolutely critical for establishing infection by M. tuberculosis, its conserved nature in all the pathogenic and nonpathogenic mycobacterial species intrigues to explore the additional functional roles in Mycobacterium species through which potent targets for drugs can be identified and developed. In the present study, we investigated the possible role of EccD3, a transmembrane protein of the ESX3 T7SS in M. smegmatis by deleting the entire eccD3 gene by efficient allelic exchange method. The preliminary investigations through the creation of knockout mutant of the eccD3 gene indicate that this secretory apparatus has an important role in maintaining the cell wall integrity which was evident from the abnormal colony morphology, lack of biofilm formation and difference in cell wall permeability., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The ESAT-6-like secretion system (ESS) of Staphylococcus aureus is assembled in the bacterial membrane from core components that promote the secretion of WXG-like proteins (EsxA, EsxB, EsxC, and EsxD) and the EssD effector. Genes encoding the ESS secretion machinery components, effector, and WXG-like proteins are located in the ess locus. Here, we identify essH , a heretofore uncharacterized gene of the ess locus, whose product is secreted via an N-terminal signal peptide into the extracellular medium of staphylococcal cultures. EssH exhibits two peptidoglycan hydrolase activities, cleaving the pentaglycine cross bridge and the amide bond of N -acetylmuramyl-l-alanine, thereby separating glycan chains and wall peptides with cleaved cross bridges. Unlike other peptidoglycan hydrolases, EssH does not promote the lysis of staphylococci. EssH residues Cys 199 and His 254 , which are conserved in other CHAP domain enzymes, are required for peptidoglycan hydrolase activity and for S. aureus ESS secretion. These data suggest that EssH and its murein hydrolase activity are required for protein secretion by the ESS pathway. IMPORTANCE Gene clusters encoding WXG-like proteins and FtsK/SpoIIIE-like P loop ATPases in Firmicutes encode type 7b secretion systems (T7bSS) for the transport of select protein substrates. The Staphylococcus aureus T7bSS assembles in the bacterial membrane and promotes the secretion of WXG-like proteins and effectors. The mechanisms whereby staphylococci extend the T7SS across the bacterial cell wall envelope are not known. Here, we show that staphylococci secrete EssH to cleave their peptidoglycan, thereby enabling T7bSS transport of proteins across the bacterial cell wall envelope., (Copyright © 2018 American Society for Microbiology.)

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum., Competing Interests: The authors have declared that no competing interests exist.

Conjugal cell-cell contact between strains of Mycobacterium smegmatis induces the esxUT transcript, which encodes the putative primary substrates of the ESAT-6 secretion system 4 (ESX-4) secretion system. This recipient response was required for conjugal transfer of chromosomal DNA from the donor strain. Here we show that the extracytoplasmic σ factor, SigM, is a cell contact-dependent activator of ESX-4 expression and is required for conjugal transfer of DNA in the recipient strain. The SigM regulon includes genes outside the seven-gene core esx4 locus that we show are also required for conjugation, and we show that some of these SigM-induced proteins likely function through ESX-4. A fluorescent reporter revealed that SigM is specifically activated in recipient cells in direct contact with donor cells. Coculture RNA-seq experiments indicated that SigM regulon induction occurred early and before transconjugants are detected. This work supports a model wherein donor contact with the recipient cell surface inactivates the transmembrane anti-SigM, thereby releasing SigM. Free SigM induces an extended ESX-4 secretion system, resulting in changes that facilitate chromosomal transfer. The contact-dependent inactivation of an extracytoplasmic σ-factor that tightly controls ESX-4 activity suggests a mechanism dedicated to detect, and appropriately respond to, external stimuli from mycobacteria., Competing Interests: The authors declare no conflict of interest.

The ESAT6-like Secretion System (ESS) of the human pathogen Staphylococcus aureus secretes heterodimeric virulence effectors such as EsxB and EsxD. To gain insights into the nature of EsxB-EsxD interaction, randomly mutated esxB generated by error-prone PCR was co-transformed together with esxD as adenylate cyclase fusion constructs into cyclase-deficient Escherichia coli, followed by reverse bacterial two-hybrid screening. Three color species were observed: dark blue, light blue, and white (no EsxB-EsxD interaction). The esxB from white colonies was subjected to standard PCR to check for gene signal, followed by SDS-PAGE for variant stability assessment. The gene coding for a stable EsxB variant that perturbed interaction with EsxD was further subjected to DNA sequencing. A single point mutation in esxB at position 157 was identified, leading to an amino acid change from asparagine to aspartic acid at position 53 in the resulting protein. Structural modeling of EsxB reveals that N53 is surface exposed. Whereas N53S substitution by site-directed mutagenesis retained heterodimerization with EsxD, N53A substitution abrogated such interaction. In addition, N53D change in EsxB did not alter interaction with EssG, another soluble component of the ESS pathway, suggesting minimal impact of the N53D substitution on EsxB stability and solubility. Taken together, these data provide new insights into the nature of EsxB-EsxD interaction and offer a systematic approach for in vivo analysis of protein-protein interactions of pathogenic bacteria in non-pathogenic hosts.