Type VII secretion system extracellular protein B targets STING to evade host anti-Staphylococcus aureus immunity.

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.

New insights into the domain of unknown function (DUF) of EccC5, the pivotal ATPase providing the secretion driving force to the ESX-5 secretion system.

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccCDUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Šresolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.

The antagonistic transcription factors, EspM and EspN, regulate the ESX-1 secretion system in M. marinum.

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.

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

ESX-3 secretion system in Mycobacterium: An overview.

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.

Three small partner proteins facilitate the type VII-dependent secretion of an antibacterial nuclease.

IMPORTANCE: Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.

Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins.

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.

Type VII secretion system and its effect on group B Streptococcus virulence in isolates obtained from newborns with early onset disease and colonized pregnant women.

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.

Mycobacterial type VII secretion systems.

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion.

A novel variant of the Listeria monocytogenes type VII secretion system EssC component is associated with an Rhs toxin.

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.

Intragranuloma Accumulation and Inflammatory Differentiation of Neutrophils Underlie Mycobacterial ESX-1-Dependent Immunopathology.

The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6C+MHCII+ monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6C+MHCII+ monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. IMPORTANCE The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.

High Bacillary Burden and the ESX-1 Type VII Secretion System Promote MHC Class I Presentation by Mycobacterium tuberculosis-Infected Macrophages to CD8 T Cells.

We used a mouse model to study how Mycobacterium tuberculosis subverts host defenses to persist in macrophages despite immune pressure. CD4 T cells can recognize macrophages infected with a single bacillus in vitro. Under identical conditions, CD8 T cells inefficiently recognize infected macrophages and fail to restrict M. tuberculosis growth, although they can inhibit M. tuberculosis growth during high-burden intracellular infection. We show that high intracellular M. tuberculosis numbers cause macrophage death, leading other macrophages to scavenge cellular debris and cross-present the TB10.4 Ag to CD8 T cells. Presentation by infected macrophages requires M. tuberculosis to have a functional ESX-1 type VII secretion system. These data indicate that phagosomal membrane damage and cell death promote MHC class I presentation of the immunodominant Ag TB10.4 by macrophages. Although this mode of Ag presentation stimulates cytokine production that we presume would be host beneficial, killing of uninfected cells could worsen immunopathology. We suggest that shifting the focus of CD8 T cell recognition to uninfected macrophages would limit the interaction of CD8 T cells with infected macrophages and impair CD8 T cell-mediated resolution of tuberculosis.

Cryo-EM reveals the membrane-binding phenomenon of EspB, a virulence factor of the mycobacterial type VII secretion system.

Mycobacterium tuberculosis (Mtb) utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. EspB, a ∼36 kDa secreted substrate of the ESX-1 apparatus, was shown to cause ESAT-6-independent host cell death. Despite the current wealth of high-resolution structural information of the ordered N-terminal domain, the mechanism of EspB-mediated virulence remains poorly characterized. Here, we document EspB interaction with phosphatidic acid (PA) and phosphatidylserine (PS) in the context of membranes, through a biophysical approach including transmission electron microscopy and cryo-EM. We were also able to show PA, PS-dependent conversion of monomers to oligomers at physiological pH. Our data suggest that EspB adheres to biological membranes with limited PA and PS. EM of yeast mitochondria with EspB indicates a mitochondrial membrane-binding property of this ESX-1 substrate. Further, we determined the 3D structures of EspB with and without PA and observed plausible stabilization of the low complexity C-terminal domain in the presence of PA. Collectively, our cryo-EM-based structural and functional studies of EspB provide further insight into the host-Mtb interaction.

Dysregulation of Mycobacterium marinum ESX-5 Secretion by Novel 1,2,4-oxadiazoles.

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.

Role of the Mycobacterium tuberculosis ESX-4 Secretion System in Heme Iron Utilization and Pore Formation by PPE Proteins.

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.

Progress on the function of Mycobacterium T7SS (ESX) secretion system.

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.

Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.

Pathogenic mycobacteria use the ESX-1 secretion system to escape the macrophage phagosome and survive infection. We demonstrated that the ESX-1 system is regulated by feedback control in Mycobacterium marinum, a nontuberculous pathogen and model for the human pathogen Mycobacterium tuberculosis. In the presence of a functional ESX-1 system, the WhiB6 transcription factor upregulates expression of ESX-1 substrate genes. In the absence of an assembled ESX-1 system, the conserved transcription factor, EspM, represses whiB6 expression by specifically binding the whiB6 promoter. Together, WhiB6 and EspM fine-tune the levels of ESX-1 substrates in response to the secretion system. The mechanisms underlying control of the ESX-1 system by EspM are unknown. Here, we conduct a structure and function analysis to investigate how EspM is regulated. Using biochemical approaches, we measured the formation of higher-order oligomers of EspM in vitro. We demonstrate that multimerization in vitro can be mediated through multiple domains of the EspM protein. Using a bacterial monohybrid system, we showed that EspM self-associates through multiple domains in Escherichia coli. Using this system, we performed a genetic screen to identify EspM variants that failed to self-associate. The screen yielded four EspM variants of interest, which we tested for activity in M. marinum. Our study revealed that the two helix-turn-helix domains are functionally distinct. Moreover, the helix bundle domain is required for wild-type multimerization in vitro. Our data support models where EspM monomers or hexamers contribute to the regulation of whiB6 expression. IMPORTANCE Pathogenic mycobacteria are bacteria that pose a large burden to human health globally. The ESX-1 secretion system is required for pathogenic mycobacteria to survive within and interact with the host. Proper function of the ESX-1 secretion system is achieved by tightly controlling the expression of secreted virulence factors, in part through transcriptional regulation. Here, we characterize the conserved transcription factor EspM, which regulates the expression of ESX-1 virulence factors. We define domains required for EspM to form multimers and bind DNA. These findings provide an initial characterization an ESX-1 transcription factor and provide insights into its mechanism of action.

Need for speed: Key driver of host cell migration varies among mycobacteria.

In this issue of Cell, Saelens et al. describe a new function for mycobacterial Type VII secretion systems: manipulation of host cell migration. They find that a substantial proportion of global TB cases arise from bacteria lacking this function, raising questions about its role in pathoadaptation of Mycobacterium tuberculosis.

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum.

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis.

Role of Klebsiella pneumoniae Type VI secretion system (T6SS) in long-term gastrointestinal colonization.

Type VI secretion systems (T6SS), recently described in hypervirulent K. pneumoniae (hvKp) strains, are involved in bacterial warfare but their role in classical clinical strains (cKp) has been little investigated. In silico analysis indicated the presence of T6SS clusters (from zero to four), irrespective of the strains origin or virulence, with a high prevalence in the K. pneumoniae species (98%). In the strain CH1157, two T6SS-apparented pathogenicity islands were detected, T6SS-1 and -2, harboring a phospholipase-encoding gene (tle1) and a potential new effector-encoding gene named tke (Type VI Klebsiella effector). Tle1 expression in Escherichia coli periplasm affected cell membrane permeability. T6SS-1 isogenic mutants colonized the highest gastrointestinal tract of mice less efficiently than their parental strain, at long term. Comparative analysis of faecal 16S sequences indicated that T6SS-1 impaired the microbiota richness and its resilience capacity. Oscillospiraceae family members could be specific competitors for the long-term gut establishment of K. pneumoniae.