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Key Points 6 kDa early secretory antigenic target (ESAT6) secretion systems (ESX; also known as type VII secretion systems) are sophisticated secretion systems that are present in a wide variety of mycobacterial and non-mycobacterial members of the phylum Actinobacteria. The ESX-1 system of Mycobacterium tuberculosis is the most well-studied ESX system, owing to its important function in virulence, which is linked — at least in part — to the ability of one of its secreted effector proteins, EsxA, to induce phagosomal rupture in host phagocytes. ESX-1 systems are also present in many non-pathogenic, rapid-growing mycobacteria, such as Mycobacterium smegmatis , in which they are involved in conjugal DNA transfer between donor and recipient strains. Interestingly, the ESX-1 effectors EspA, EspC and EspD, which are present in slow-growing, pathogenic mycobacteria, are absent from these non-pathogenic species. Besides ESX-1, M. tuberculosis , which causes tuberculosis, has four additional ESX systems: ESX-3 is involved in iron acquisition; ESX-5 is involved in the secretion of members from two large mycobacterial protein families, named PE and PPE according to their Pro-Glu and Pro-Pro-Glu amino-terminal motifs; and ESX-2 and ESX-4 are systems for which the functions are currently unknown. ESX systems are thought to have evolved from ESX-4 or ESX-4-like systems by gene duplication and diversification, as well as plasmid-mediated horizontal gene transfer. ESX-like systems are secretion systems that are similar to mycobacterial ESX systems but that are found in Gram-positive bacteria in the phylum Firmicutes, rather than in Actinobacteria. Similarly to ESX systems, ESX-like systems contain Esx proteins that have a highly conserved WXG motif, contain an Ftsk–SpoIIIE-like ATPase, and, in some cases (for example, in Staphylococcus aureus ), can be involved in pathogenicity. Mycobacteria use ESX systems to secrete substrates across their cell envelopes. In this Review, Brosch and colleagues describe the roles of ESX systems in host–pathogen interactions and consider how studies of ESX systems might inform vaccine design and therapy development. Mycobacterium tuberculosis uses sophisticated secretion systems, named 6 kDa early secretory antigenic target (ESAT6) protein family secretion (ESX) systems (also known as type VII secretion systems), to export a set of effector proteins that helps the pathogen to resist or evade the host immune response. Since the discovery of the esx loci during the M. tuberculosis H37Rv genome project, structural biology, cell biology and evolutionary analyses have advanced our knowledge of the function of these systems. In this Review, we highlight the intriguing roles that these studies have revealed for ESX systems in bacterial survival and pathogenicity during infection with M. tuberculosis . Furthermore, we discuss the diversity of ESX systems that has been described among mycobacteria and selected non-mycobacterial species. Finally, we consider how our knowledge of ESX systems might be applied to the development of novel strategies for the treatment and prevention of disease.

Staphylococcus aureus is a leading cause of infections worldwide. S. aureus utilizes a specialized type VIIb secretion system (T7SSb) to persist in the infected host tissues as well as target competitor bacteria to establish its niche. T7SSb assembles into a multiprotein translocation complex and facilitates secretion of a set of small proteins and larger polymorphic toxins across the cytosolic membrane. Beyond the membrane, secreted proteins were thought to diffuse through the thick yet porous cell wall and release into the environment. Here, we demonstrate for the first time that S. aureus T7SSb extends across the cell wall via its EsaA subunit. Furthermore, accommodation of EsaA within the cell wall requires an associated cell wall hydrolase EssH and is essential for protein secretion via T7SSb. Thus, our findings provide a mechanistic insight for a coordinated cell wall processing and T7SSb assembly to support specialized protein secretion in S. aureus .

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.

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 EccB3, EccC3, and EccE3 and two copies of the EccD3 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.

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

Communal bacterial processes require intercellular communication mediated by secretion systems to coordinate appropriate molecular responses. Intercellular communication has not been described previously in mycobacteria. Here we show that the ESX secretion-system family member ESX-4 is essential for conjugal recipient activity in Mycobacterium smegmatis. Transcription of esx4 genes in the recipient requires coculture with a donor strain and a functional ESX-1 apparatus in the recipient. Conversely, mutation of the donor ESX-1 apparatus amplifies the esx4 transcriptional response in the recipient. The effect of ESX-1 on esx4 transcription correlates with conjugal DNA transfer efficiencies. Our data show that intercellular communication via ESX-1 controls the expression of its evolutionary progenitor, ESX-4, to promote conjugation between mycobacteria.

Type VII secretion systems (T7SS) are increasingly recognized as an indispensable secretion system for Gram-positive bacteria, mediating processes vital for bacterial survival and pathogenesis. This work reveals a previously unrecognized mode of active iron acquisition mediated by T7SS, which not only boosts oxidative stress resistance in Corynebacterium glutamicum but also provides a competitive edge under nutrient-limited conditions. ExsI homologs in Mycobacterium smegmatis overcome calprotectin-mediated iron withholding, suggesting that T7SS-driven iron acquisition extends to immune evasion. These uncovered previously unreported functions of T7SS emphasize the indispensable importance of T7SS in bacterial physiology, enhancing our understanding of T7SS.

Background Type VII protein secretion (T7SS) is a specialised system for excreting extracellular proteins across bacterial cell membranes and has been associated with virulence in Staphylococcus aureus . The genetic diversity of the ess locus, which encodes the T7SS, and the functions of proteins encoded within it are poorly understood. Results We used whole genome sequence data from 153 isolates representative of the diversity of the species to investigate the genetic variability of T7SS across S. aureus . The ess loci were found to comprise of four distinct modules based on gene content and relative conservation. Modules 1 and 4, comprising of the 5’ and 3’ modules of the ess locus, contained the most conserved clusters of genes across the species. Module 1 contained genes encoding the secreted protein EsxA, and the EsaAB and EssAB components of the T7SS machinery, and Module 4 contained two functionally uncharacterized conserved membrane proteins. Across the species four variants of Module 2 were identified containing the essC gene, each of which was associated with a specific group of downstream genes. The most diverse module of the ess locus was Module 3 comprising a highly variable arrangement of hypothetical proteins. RNA-Seq was performed on representatives of the four Module 2 variants and demonstrated strain-specific differences in the levels of transcription in the conserved Module 1 components and transcriptional linkage Module 2, and provided evidence of the expression of genes the variable regions of the ess loci. Conclusions The ess locus of S. aureus exhibits modularity and organisational variation across the species and transcriptional variation. In silico analysis of ess loci encoded hypothetical proteins identified potential novel secreted substrates for the T7SS. The considerable variety in operon arrangement between otherwise closely related isolates provides strong evidence for recombination at this locus. Comparison of these recombination regions with each other, and with the genomes of other Staphylococcal species, failed to identify evidence of intra- and inter-species recombination, however the analysis identified a novel T7SS in another pathogenic staphylococci, Staphylococcus lugdunensis .

Abstract Macrophages are first-line responders against microbes. The success of Mycobacterium tuberculosis (Mtb) rests upon its ability to convert these antimicrobial cells into a permissive cellular niche. This is a remarkable accomplishment, as the antimicrobial arsenal of macrophages is extensive. Normally bacteria are delivered to an acidic, degradative lysosome through one of several trafficking pathways, including LC3-associated phagocytosis (LAP) and autophagy. Once phagocytozed, the bacilli are subjected to reactive oxygen and nitrogen species, and they induce the expression of proinflammatory cytokines, which serve to augment host responses. However, Mtb hijacks these host defense mechanisms, manipulating host cellular trafficking, innate immune responses, and cell death pathways to its benefit. The complex series of measures and countermeasures between host and pathogen ultimately determines the outcome of infection. In this review, we focus on the diverse effectors that Mtb uses in its multipronged effort to subvert the innate immune responses of macrophages. We highlight recent advances in understanding the molecular interface of the Mtb–macrophage interaction. The authors focus on the diverse effectors that Mycobacterium tuberculosis uses in its multi-pronged effort to subvert the innate immune responses of macrophages.

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.