Bacterial secretion system | EPFL Graph Search

Bacterial secretion systems are protein complexes present on the cell membranes of bacteria for secretion of substances. Specifically, they are the cellular devices used by pathogenic bacteria to secrete their virulence factors (mainly of proteins) to invade the host cells. They can be classified into different types based on their specific structure, composition and activity. Generally, proteins can be secreted through two different processes. One process is a one-step mechanism in which proteins from the cytoplasm of bacteria are transported and delivered directly through the cell membrane into the host cell. Another involves a two-step activity in which the proteins are first transported out of the inner cell membrane, then deposited in the periplasm, and finally through the outer cell membrane into the host cell. These major differences can be distinguished between Gram-negative diderm bacteria and Gram-positive monoderm bacteria. But the classification is by no means clear and c

Functional characterization of the virulence determinant ESX-1 from Mycobacterium tuberculosis

Paloma Julia Soler Arnedo

Tuberculosis (TB) is a chronic infectious disease that mainly affects the lungs and causes extensive human morbidity and mortality. It results from infection with Mycobacterium tuberculosis, a slow-growing intracellular pathogen that can replicate and survive inside macrophages. M. tuberculosis relies on the specialised ESX-1 secretion system to export virulence factors needed for intracellular spread and pathogenesis. The ESX-1 apparatus is a multi-subunit nanomachine composed of ~20 polypeptides including membrane proteins, ATPases, proteases, chaperones and substrates. Although many of the individual components of this secretion system have been characterised, the overall mechanism underlying ESX-1 secretion is still far from clear. To obtain a more comprehensive picture of the functioning of the ESX-1 apparatus, we have studied various components which were largely unexplored in M. tuberculosis. The structural component EccE1, the ESX-1 specific protein EspL and the transcriptional regulator WhiB6 have been investigated in this thesis using an integrative approach involving genetics, biochemistry, proteomics and microscopy. We have demonstrated that EccE1 is a membrane- and cell-wall associated protein critical for secretion of ESX-1 substrates and M. tuberculosis-mediated cell lysis. Deletion of eccE1 from the chromosome severely compromised secretion of EsxA, EsxB, EspA and EspC but not EspB. Localization studies using a florescent-fusion protein showed that EccE1 localises to the poles of M. tuberculosis in the presence of an active ESX-1 system. Our study also shows that EspL is a cytosolic protein needed for stabilising EspE, EspF and EspH, suggesting that it acts as a specific chaperone of the ESX-1 secretion system. Moreover, EspL was shown to interact with EspD and to be important for the secretion of ESX-1 substrates. Lack of EspL resulted in a growth defect ex vivo, loss of cytotoxicity and reduction of innate cytokine production demonstrating its critical role in M. tuberculosis virulence. Analysis of the transcriptional response revealed that the only gene deregulated in the absence of espL was whiB6, encoding a transcriptional factor that positively controls ESX-1 genes. To explore the role of this regulator in M. tuberculosis virulence, we generated a deletion mutant of whiB6 and discovered that its loss resulted in severe reduction of cytotoxicity ex vivo. Overall this investigation improves our current understanding of the ESX-1 secretion system and of the molecular basis of M. tuberculosis virulence. The increased knowledge of the complex interactions between the pathogen and the human host will hopefully translate into new strategies to control the spread of TB.

EPFL2018

Structural and functional characterization of the mycobacterial ESX-1 secretion system

Ye Lou

Human tuberculosis (TB) is a frequently fatal lung disease mostly caused by Mycobacterium tuberculosis (M. tb), a slow-growing intracellular pathogen. M. tb utilizes the ESX-1 secretion system, classified as type VII, to export the virulence factors needed for host infection and pathogenesis. A functional ESX-1 pathway largely involves the proteins encoded by two gene clusters, esx-1 and espACD. These proteins are diverse in functions, such as cytolysis, immunogenicity, pore forming, stabilization and energization. Although many of the ESX-1 associated proteins were characterized individually, the overall mechanism of ESX-1 secretion is still far from clear. The main objective of my thesis is to better understand the structure and function of the ESX-1 secretion system in M. tb. To achieve this, an integrated approach which combines biochemistry, biophysics and genetics was applied. The experimental results are organized into three parts, in which I investigated EspC, EccCb1 and the effect of calcium on ESX-1 secretion. Firstly, the focus of this thesis is EspC, a secreted protein encoded in the espACD operon which is not linked to the esx-1 locus, but mediates ESX-1-associated virulence. Based on what I observed from a series of experiments, we propose a novel working model of the ESX-1 apparatus, in which EspC interacts with EspA in the cytosol and then translocates across the membrane to assemble into a channel in the outer membrane and spans the capsular layer of M. tb. This model could explain the mechanism by which the major virulence factors EsxA and EsxB (also named ESAT-6 and CFP-10) rely on EspC for secretion. Moreover, EspC is a potential outer membrane pore protein which has not been previously identified in M. tb. Secondly, the putative ATPase EccCb1 is another protein that was studied. EccCb1 is predicted as an ATPase that targets the EsxA/EsxB heterodimer during translocation. EccCb1 was overexpressed and purified in the form of a NusA- EccCb1 fusion protein due to its low stability. The protein was characterized as a hexamer with an ATPase activity. This result confirms that EccCb1 belongs to the FtsK/SpoIIIE ATPase family. Thirdly, we observed that ESX-1 secretion can be inhibited by a high concentration of calcium (~500 ÎŒM) in the culture medium. Moreover, bacterial persistence, rather than growth rate, decreased with the elevated calcium. RNA- seq was performed to evaluate the changes of global gene expression of M. tb in the presence of high calcium levels. Surprisingly, calcium dramatically repressed hypoxia response genes in the DevRS regulon, leading to reduced persistence. I also include a section that describes the additional work I was involved with, in which we investigated the activation of cGAS-dependent host response by ESX-1. Taken together, these findings provide new insights into the structural and functional aspects of ESX-1 secretion system, and will contribute to discovery of ESX-1 inhibitors with potential applications in TB therapy.

EPFL2016

Structure and properties of the C-terminal beta-helical domain of VgrG protein from Escherichia coli O157

Petr Leiman

The bacterial Type 6 secretion system (T6SS) translocates protein toxins (also called effectors) from the cytosol of a T6SS-carrying cell to a target cell by a syringe-like supramolecular complex resembling a contractile tail of bacteriophages. Valine-glycine repeat protein G (VgrG) proteins, which are the homologues of the gp27-gp5 (gene product) cell puncturing complex of bacteriophage T4, are considered to be located at the attacking tip of the bacterial T6SS apparatus. Here, we over-expressed six VgrG proteins from pathogenic Escherichia coli O157 and CFT073 strains. Purified VgrG1 of E. coli O157 and c3393 of E. coli CFT073 form trimer in solution and are rich in beta-structure. We also solved the crystal structure of a trypsin-resistant C-terminal fragment of E. coli O157 VgrG1 (VgrG1C(G561)) at 1.95 A resolution. VgrG1C(G561) forms a three-stranded antiparallel beta-helix which is structurally similar to the beta-helix domain of the central spike protein (gp138) of phi92 phage, indicating a possible evolutional relationship. Comparison of four different three-stranded beta-helix proteins shows how their amino acid composition determines the protein fold.

Oxford University Press2014