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For many gram-positive pathogens, conjugative plasmid transfer is an important means of spreading antibiotic resistance . Therefore, the search for alternative treatments to fight and prevent infections caused by these bacteria has become of major interest. In the present study, we evaluated the protein TraM, from the conjugative plasmid pIP501, as a potential vaccine candidate. Anti-TraM antiserum mediated in vitro opsonophagocytic killing of the strain harboring the pIP501 plasmid and also proved to be cross-reactive against other clinically relevant enterococcal and staphylococcal strains. Specificity of antibodies toward TraM was confirmed by results of an opsonophagocytic inhibition assay and Western blot. In addition, conjugative transfer experiments proved that TraM is essential for the transfer of pIP501. Finally, immunization with either TraM or anti-TraM antiserum reduced significantly the colony counts in mice livers, demonstrating that TraM is a promising vaccine candidate against enterococci and other gram-positive pathogens. [ABSTRACT FROM AUTHOR]

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecafis and Enterococcus cium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for piP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring piP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGATMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchito- hexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and b degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosyla Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope. [ABSTRACT FROM AUTHOR]