Caractérisation biochimique, structurale et inhibition du système de sécrétion de type IV par l’étude des protéines VirB8

In all organisms, secretion systems mediate the passage of macromolecules across cellular membranes. Gram-negative bacteria use type IV secretion systems (T4SS) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis. In chapter 2, we studied the structure and function of TraE, a homolog of VirB8 from pKM101, that is an essential core component of all T4SS. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs, but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the BTH and crosslinking assays showed that TraE and its homologs from Agrobacterium, Brucella and H. pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity of the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, in silico docking predicted that they bind to a structurally conserved surface groove of the protein and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites and these can be exploited for the design of specific inhibitors of T4SS function. The increasing prevalence of antimicrobial resistance is a problem of global importance. Novel strategies are urgently needed to understand and inhibit antimicrobial resistance gene transmission that is mechanistically related to bacterial virulence functions. The conjugative transfer of plasmids by T4SSs is a major contributor to antimicrobial resistance gene transfer. In chapter 3, we present a structure-based strategy to identify inhibitors of T4SS-mediated bacterial conjugation. Using differential scanning fluorimetry we screened a fragment library and identified molecules that bind the essential TraE protein of the plasmid pKM101 conjugation machinery. Co-crystallization revealed that fragments bind two alternative sites of iii the protein and one of them is a novel inhibitor binding site. Based on the structural information on fragment binding we designed novel small molecules that have improved binding affinity. These molecules inhibit the dimerization of TraE, bind to both inhibitor binding sites on TraE and inhibit the conjugative transfer of plasmid pKM101. The strategy presented here is generally applicable for the structure-based design of inhibitors of antimicrobial resistance gene transfer and of bacterial virulence. X-ray crystallographic and electron microscopic analyses have increasingly provided structural information on individual T4SS components and on the entire complex. But as of now, relatively little information has been available on the membrane-bound T4SS components. The overproduction and purification of membrane proteins is intrinsically difficult making their analysis challenging. In chapter 4, we show that the membrane-bound full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high molecular mass complex that is distinct from the previously characterized periplasmic portion of the protein. Full-length TraE was extracted from the membranes with detergents and analysis by size- exclusion chromatography, crosslinking and the combination of SEC with multi-angle light scattering show that it forms a high molecular mass complex. Electron microscopy and small- angle X-ray scattering analysis demonstrate that full-length TraE assembles into a hexameric complex with a central pore. We also show by crosslinking, SEC and electron microscopy that TraE binds to the TraD protein. Our results suggest that TraE and TraD form a pore at the core of the secretion system contributing to substrate translocation. This study, which combined several disciplines, including biochemistry, structural biology and chemical biology, underscores the interest and significance of a multidisciplinary approach.

Keyphrases: plasmid, conjugation, type IV secretion system, protein-protein interaction, fragment-based screening, drug design, antimicrobial resistance, VirB8-like, VirB6-like, membrane pore