Biology subjects

Houben, E. N. G.

Publications and source records attributed to Houben, E. N. G..

4 recordsLinked to original sources

bioRxiv2022

Reconstitution of a minimal ESX-5 type VII secretion systems uncovers the essential role of the PPE proteins in secretion

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 a first full overview of the role of each esx-5 gene in T7SS functionality.

microbiology↗

bioRxiv2022

The ESX-1 substrate PPE68 has a key function in the 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 as folded heterodimers. Investigations into the functions of these substrates is problematic, because of the intricate network of co-dependent secretion between several ESX-1 substrates. Here, we describe that the ESX-1 substrate PPE68 is essential for secretion of the highly immunogenic substrate 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 is not required for the secretion of PPE68, revealing hierarchy in co-dependent secretion. Remarkably, the final ten residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and 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 co-dependence. ImportanceMycobacterium tuberculosis use type VII secretion systems (T7SSs) to secrete proteins across its uniquely hydrophobic diderm cell envelope. A T7SS subtype, called ESX-1, is one of its most important virulence determinants by mediating intracellular survival through 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 co-dependent secretion between ESX-1 substrates. Here, we provide mechanistic insight into the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates, using the model organism Mycobacterium marinum. Unravelling the mechanism of co-dependent 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 this significant human pathogen.

microbiology↗

bioRxiv2020

Structure and dynamics of the ESX-5 type VII secretion system of Mycobacterium tuberculosis

Mycobacterium tuberculosis causes one of the most important infectious diseases in humans, leading to 1.5 million deaths every year. Specialized protein transport systems, called type VII secretion systems (T7SSs), are central for its virulence, but also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Here we present the first structure of an intact T7SS inner membrane complex of M. tuberculosis. We show how the 2.32 MDa, 165 transmembrane helices-containing ESX-5 assembly is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber formed by three EccB5 dimers, with the proteolytic sites facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, highlighting the importance of this component for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three four-transmembrane helix bundles, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations, likely reflecting different secretion states. Our work suggests a novel mechanism of protein transport and provides a structural scaffold to aid drug development against the major human pathogen.

microbiology↗

bioRxiv2020

Rerouting of an Esx substrate pair from the ESX-1 type VII secretion system to ESX-5 by modifying a PE/PPE substrate pair

Type VII secretion systems (T7SSs) secrete a wide range of extracellular proteins that play important roles in bacterial viability and in host-pathogen interactions of pathogenic mycobacteria. There are five subtypes of mycobacterial T7SSs, called ESX-1 to ESX-5, and four classes of T7SS substrates, namely the Esx, PE, PPE and Esp proteins. At least some of these substrates are secreted as heterodimers. The ESX systems mediate the secretion of specific members of the Esx, PE and PPE proteins, raising the question how these substrates are recognized in a system-specific fashion. PE/PPE heterodimers interact with their cognate EspG chaperones, which recently has been shown to determine their designated secretion pathway. Both structural and pulldown analysis suggest that EspG is unable to interact with Esx proteins and therefore the determining factor for system-specificity of these substrates remains unknown. In this study, we have investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 (MMAR_0187/MMAR _0188) in Mycobacterium marinum. While this substrate pair was hardly secreted when ectopically expressed, secretion was observed when EsxB_1/EsxA_1 was co-expressed together with PE35/PPE68_1 (MMAR_0185/MMAR_0186), which are encoded by the same operon. Surprisingly, co-expressing 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 co-secretion of EsxB_1/EsxA_1 via ESX-5. Our data suggest a secretion model in which PE35/PPE68_1 is a determinant factor for the system-specific secretion of EsxB_1/EsxA_1.

molecular biology↗