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.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

Structure of a tripartite protein complex that targets toxins to the type VII secretion system.

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.

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.

Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.

Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria.

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.

Characterization of TelE, a T7SS LXG Effector Exhibiting a Conserved C-Terminal Glycine Zipper Motif Required for Toxicity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the type VIIb secretion system (T7SSb) machinery is uniquely present in SGG in two different arrangements. SGG UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by this secretion system, we inactivated the essC gene encoding the motor of this machinery. A comparison of the proteins secreted by UCN34 wild type and its isogenic ΔessC mutant revealed six T7SSb effector proteins, including the expected WXG effector EsxA and three LXG-containing proteins. In this work, we characterized an LXG-family toxin named herein TelE promoting the loss of membrane integrity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C terminus were identified in different SGG isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE membrane destabilization activity. TelE activity was antagonized by a small protein TipE belonging to the DUF5085 family. Overall, we report herein a unique SGG T7SSb effector exhibiting a toxic activity against nonimmune bacteria. IMPORTANCE In this study, 38 clinical isolates of Streptococcus gallolyticus subsp. gallolyticus (SGG) were sequenced and a genetic locus encoding the type VIIb secretion system (T7SSb) was found conserved and absent from 16 genomes of the closely related S. gallolyticus subsp. pasteurianus (SGP). The T7SSb is a bona fide pathogenicity island. Here, we report that the model organism SGG strain UCN34 secretes six T7SSb effectors. One of the six effectors named TelE displayed a strong toxicity when overexpressed in Escherichia coli. Our results indicate that TelE is probably a pore-forming toxin whose activity can be antagonized by a specific immunity protein named TipE. Overall, we report a unique toxin-immunity protein pair and our data expand the range of effectors secreted through T7SSb.

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.

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.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract.

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.

Plant commensal type VII secretion system causes iron leakage from roots to promote colonization.

Competition for iron is an important factor for microbial niche establishment in the rhizosphere. Pathogenic and beneficial symbiotic bacteria use various secretion systems to interact with their hosts and acquire limited resources from the environment. Bacillus spp. are important plant commensals that encode a type VII secretion system (T7SS). However, the function of this secretion system in rhizobacteria-plant interactions is unclear. Here we use the beneficial rhizobacterium Bacillus velezensis SQR9 to show that the T7SS and the major secreted protein YukE are critical for root colonization. In planta experiments and liposome-based experiments demonstrate that secreted YukE inserts into the plant plasma membrane and causes root iron leakage in the early stage of inoculation. The increased availability of iron promotes root colonization by SQR9. Overall, our work reveals a previously undescribed role of the T7SS in a beneficial rhizobacterium to promote colonization and thus plant-microbe interactions.

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.

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.

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.

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.

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.

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.