Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year
. Specialized protein transport systems-known as ...type VII secretion systems (T7SSs)-are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope
. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP
protease. A trimer of MycP
caps a central periplasmic dome-like chamber that is formed by three EccB
dimers, with the proteolytic sites of MycP
facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP
show disruption of the EccB
periplasmic assembly and increased flexibility, which highlights the importance of MycP
for complex integrity. Beneath the EccB
-MycP
chamber, dimers of the EccC
ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC
domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.
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.
Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their complex cell envelope. Pathogenic mycobacteria, such as the notorious pathogen Mycobacterium tuberculosis, have up ...to five of these secretion systems, named ESX-1 to ESX-5. At least three of these secretion systems are essential for mycobacterial virulence and/or viability. Elucidating T7S is therefore essential to understand the success of M. tuberculosis and other pathogenic mycobacteria as pathogens, and could be instrumental to identify novel targets for drug- and vaccine-development. Recently, significant progress has been achieved in the identification of T7S substrates and a general secretion motif. In addition, a start has been made with unraveling the mechanism of secretion and the structural analysis of the different subunits. This review summarizes these recent findings, which are incorporated in a working model of this complex machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
•There are up to five different T7S systems in Mycobacteria.•The secreted substrates are usually secreted as folded dimers.•Substrates have a similar bundle of 4 alpha-helices followed by a secretion motif.•The core of the secretion machinery is composed of four conserved proteins.•A dedicated protease has a dual role in T7S.
Summary
Pathogenic mycobacteria require type VII secretion (T7S) systems to transport virulence factors across their complex cell envelope. These bacteria have up to five of these systems, termed ...ESX‐1 to ESX‐5. Here, we show that ESX‐5 of Mycobacterium tuberculosis mediates the secretion of EsxN, PPE and PE_PGRS proteins, indicating that ESX‐5 is a major secretion pathway in this important pathogen. Using the ESX‐5 system of Mycobacterium marinum and Mycobacterium bovis BCG as a model, we have purified and analysed the T7S membrane complex under native conditions. blue native‐PAGE and immunoprecipitation experiments showed that the ESX‐5 membrane complex of both species has a size of ∼ 1500 kDa and is composed of four conserved membrane proteins, i.e. EccB5, EccC5, EccD5 and EccE5. Subsequent limited proteolysis suggests that EccC5 and EccE5 mostly reside on the periphery of the complex. We also observed that EccC5 and EccD5 expression is essential for the formation of a stable membrane complex. These are the first data on a T7S membrane complex and, given the high conservation of its components, our data can likely be generalized to most mycobacterial T7S systems.
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.
The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of ...the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.
...we would like to emphasize that the introduction of a uniform gene nomenclature for other secretion systems in Gram-negative bacteria (type II, type III) has facilitated comparative analysis of ...these systems. Amongst the different topology prediction programs that were used (TMHMM Server v. 2.0, MEMSAT3, Philius, SCAMPI, HMMTOP and Phobius) MEMSAT3 gave the correct prediction for the highest number of Ecc membrane proteins. ...only the topology prediction results of TMHMM (used on the TubercuList server) and MEMSAT3 are shown.
An effective host immune response to mycobacterial infection must control pathogen dissemination without inducing immunopathology. Constitutive overexpression of mycobacterial heat shock protein ...(myHsp70) is associated with impaired bacterial persistence, but the immune-mediated mechanisms are unknown. We found that myHsp70, in addition to enhancing antigen delivery to human dendritic cells, signaled through the CCR5 chemokine receptor, promoting dendritic cell aggregation, immune synapse formation between dendritic cells and T cells, and the generation of effector immune responses. Thus, CCR5 acts as a pattern-recognition receptor for myHsp70, which may have implications for both the pathophysiology of tuberculosis and the use of myHsps in tumor-directed immunotherapy.
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 EccC5, possessing four (putative) nucleotide‐binding domains (NBDs), is responsible for this. By creating M. marinum‐M. tuberculosis EccC5 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 EccC5. This region connects NBD1 and NBD2 of EccC5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX‐5 substrate EsxN, predicted to bind to NBD3 on EccC5, 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 EccC5.
One of the major virulence factors of Mycobacterium tuberculosis and other pathogenic mycobacteria are the type VII secretion systems. Here, we provide an important insight into the mechanism of substrate recognition by these systems by identifying a putative second substrate recognition site on the central type VII secretion membrane ATPase EccC.
Chaperones are central players in maintaining the proteostasis in all living cells. Besides highly conserved generic chaperones that assist protein folding and assembly in the cytosol, additional ...more specific chaperones have evolved to ensure the successful trafficking of proteins with extra-cytoplasmic locations. Associated with the distinctive secretion systems present in bacteria, different dedicated chaperones have been described that not only keep secretory proteins in a translocation competent state, but often are also involved in substrate targeting to the specific translocation channel. Recently, a new class of such chaperones has been identified that are involved in the specific recognition of substrates transported via the type VII secretion pathway in mycobacteria. In this minireview, we provide an overview of the different bacterial chaperones with a focus on their roles in protein secretion and will discuss in detail the roles of mycobacterial type VII secretion chaperones in substrate recognition and targeting.
PDF
