Biology subjects

Bunduc, C. M.

Publications and source records attributed to Bunduc, C. M..

3 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↗

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↗

bioRxiv2019

Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5

Type VII secretion systems (T7SSs) are used by mycobacteria to translocate a wide range of effector proteins across their diderm cell envelope. These systems, also known as ESX systems, have crucial roles for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We previously observed species-specificity in the secretion of the PE_PGRS proteins by the ESX-5 system [1], in that the M. tuberculosis ESX-5 system was unable to fully complement an M. marinum esx-5 mutant. In this study, we established that the responsible factor for this is the central membrane ATPase EccC5, which has three nucleotide binding domains (NBDs). By creating chimeric M. marinum/M. tuberculosis EccC5 constructs, we observed that PE_PGRS secretion is mediated only in the presence of an EccC5 containing the cognate linker 2, irrespective of the origin of the EccC5 backbone. This region is responsible for linking the first two NBDs and for keeping the first NBD in an inhibited state. Notably, this region is disordered in a EccC crystal structure and is particularly extended in EccC proteins of the different ESX-5 systems. These results indicate that this region is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC5.

microbiology↗