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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
...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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. ...Mycobacterial type VII secretion systems consist of five subtypes, ESX-1–5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum. Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.
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.
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 EspG
chaperone. Interfering with the binding of EspG
to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast,
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.
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.
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