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In all organisms, secretion systems mediate the passage of macromolecules across cellular membranes. The bacterial type IV secretion system (T4SS) family can be divided into three functional groups. First, as typified by the Brucella suis system, T4SSs deliver effector macromolecules into eukaryotic cells during the course of infection. Second, in some Gram-negative bacteria, such as in Helicobacter pylori (ComB system), T4SSs mediate DNA uptake from and release into the extracellular environment. Thirdly, as in the IncN plasmid pKM101, T4SSs can mediate the conjugative transfer of plasmid DNA or transposons into a wide range of bacterial species. This conjugation phenomenon contributes to the spread of antibiotic resistance genes among pathogenic bacteria, leading to the emergence of multidrug-resistant pathogenic strains. TraE of the IncN plasmid pKM101 belongs to the VirB8 family of proteins, an essential component of most T4SSs that form functional dimmers in the T4SS core. Here, we present the X-ray crystallographic structure of the periplasmic domain of TraE at 2.4 Å resolution. The structure shows many similarities to the known VirB8-like protein structures from Brucella suis [1] and Agrobacterium tumefaciens [2]. However, the nature and the number of residues implicated in the dimerization interface differ considerably from those in the TraE structure [2]. Similar to other VirB8 homologs we have shown by analytical gel filtration that there is a concentration dependant equilibrium between monomeric and dimeric forms of TraE. Moreover, using a bacterial two-hybrid assay, in vivo dimerization has been demonstrated with full-length TraE and key residues for dimerization were identified by site-directed mutagenesis. Our work adds novel insights into the growing body of knowledge on VirB8-like proteins and it will inform future strategies aimed at developing inhibitors of TraE protein interactions and of plasmid transfer.
abstracts
ABSTRACT: Bacterial T4SS are complexes, constituted of 8 to 12 conserved proteins, used by many gram-negative bacteria for the translocation of proteins and DNA-protein complexes as well as for the transportation of DNA-protein complexes across their cell envelope. T4SS are excellent model targets for the development of antivirulence drugs as it is an essential virulence factor for many bacterial pathogens, such as Brucella. Antivirulence drugs that deprive the pathogen of its essential virulence factor, the T4SS, would constitute alternatives to or enhancements of current antibiotic treatment. VirB8, a conserved assembly factor in T4SS forms dimers that are very important for T4SS function in these pathogens. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors but also a possible target for drugs that could target its protein-protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.
