Structural basis of Helicobacter pylori type IV secretion system – SUBSIST

Bacterial pathogens have evolved dedicated strategies to colonise organs and eventually to counter-attack and subvert host immune defence systems. Type IV secretion systems (T4SS) are macromolecular devices that bacteria use to transport various macromolecules including protein or nucleic acid/protein complexes across the cell envelope. These systems are remarkably versatile and are involved in bacterial conjugation and DNA uptake or release from and to the extracellular milieu. They are also used by many bacterial pathogens,to deliver protein effectors into the infected cells.
Helicobacter pylori is a Gram-negative bacterium that colonises the human stomach in half of the world population. It is estimated that 20% of individuals infected during their childhood will develop peptic ulcer disease, while gastric neoplasia will develop in 1-2% of infected individuals. For these reasons, H. pylori was defined as a group 1 carcinogen by World Health Organization (WHO). H. pylori strains isolated from patients with the most severe diseases produce the oncoprotein CagA, a unique protein encoded by the cagA gene located on the cag pathogenicity island (cagPAI). The cagPAI encodes for a T4SS (cagT4SS), a molecular device used by the bacteria to deliver CagA into the host cell. After translocation, CagA is tyrosine-phosphorylated by host Src kinases and hijacks the signalling system of the cell. This leads to cell proliferation and eventually tumour development. CagA is therefore the major determinant of cancer development during H. pylori infection and is considered the paradigm of “bacterial carcinogenesis”. The molecular mechanism of CagA injection by the cagT4SS is still poorly understood. Compared to the prototypitical T4SS, the cag system is highly complex and its structure and composition is largely unknown.
The cagT4SS of H. pylori strain 26695 is composed of 28 proteins. By sequence comparison with the prototypical VirB/D proteins from the Agrobacterium tumefaciens T4SS, some putative homologues have been identified, with most having only very limited sequence similarity, and the remaining 17 proteins being unique to H. pylori. The cagT4SS can be separated into three structural entities based on its resemblance to the A. tumefaciens T4SS: 1) a cytoplasmic complex where processing of the substrate (CagA) an its docking to the cagT4SS is taking place, 2) a core complex in the periplasmic space that might act as a channel and 3) an external appendage named pilus that interact with the host-cell. Three proteins CagI, CagL and CagH form a complex in vivo and are essential for the biogenesis of the cagT4SS pilus and CagA injection. However the structure and the function of this complex in the cagT4SS context are still unknown.
The objective of the proposed research program is to gain insight into structure and function of the CagI, CagL and CagH proteins of the cagT4SS. Our research initiative builds upon previous work in our research groups that has led to important preliminary results, including the characterisation of protein-protein interactions in vivo and in vitro and preliminary structural studies. Our short-term goal is to determine the structure and functions of the CagI/CagL/CagH protein complexes involved in the cagT4SS pilus biogenesis and define the interaction network between these pilus-associated proteins and the other sub-complexes of the cagT4SS machinery. The project involves three partners (two groups from the CNRS and one external partner from Germany) and connects structural studies (X-ray crystallography and Electron Microscopy (EM)) with interaction studies, microbiology and cellular assays. Our groups have a history of successful collaboration and our expertise is genuinely complementary. The long-term objectives are to better understand the molecular mechanisms of bacterial infection and to provide new therapeutic strategies, urgently needed to combat them.