Membrane proteins of Pasteurella multocida : roles in immunity & disease

The cell surface protects Gram-negative bacteria against a range of harsh environments and is critical for interaction of the bacterium the host cells and tissues. The outer membrane (OM) functions as a selective barrier that prevents the entry of many toxic molecules into the cell, a property that is crucial for bacterial survival in many environments. At the same time, the embedded proteins in the outer membrane fulfil a number of tasks that are crucial to the bacterial cell, such as nutrient uptake, transport of various molecules in and out of the cell, and interaction with host tissues. Pathogens have evolved different types of transport systems that contribute to their survival and dissemination within the host. The pumps that effect efflux, which include members of the TolC family, typically export several unrelated substances, including antibiotics, organic solvents, and bile produced by the host. While much data exist vis-a-vis the underlying mechanisms of multidrug efflux pumps in bacteria, in Pasteurella multocida this remains unknown. In this study, we characterised two outer membrane proteins, encoded by the genes pm0527 and pm1980, predicted to be TolC homologues in P. multocida. Compared to the wild type, the pm0527 mutant showed up to a 512-fold increase in susceptibility to a range of antibiotics and other chemical agents. Complementation of the mutant with an intact pm0527 gene restored the resistance to gentamicin, novobiocin and numerous other compounds. The pm1980 showed a 64-fold increase in susceptibility to rifampin. Together, these findings provide strong evidence that PM0527 and PM1980 function as components of multidrug efflux pumps that contribute to the intrinsic resistance of P. multocida to a broad range of structurally unrelated antibacterial agents. Methionine is an essential amino acid for all living organisms. However, in bacteria no prior evidence has been reported to substantiate this claim in vivo. Here, we characterised a predicted methionine-binding lipoprotein, PlpB, and investigated its role in the pathogenesis of P. multocida. Inactivation of the plpB gene in P. multocida resulted in full attenuation in chickens, as assessed by both direct challenge and competitive in vivo growth assays. Virulence was restored by complementation with an intact plpB gene. In vitro biochemical analyses using radiolabelled amino acids demonstrated that soluble recombinant PlpB can bind both L- and D-methionine. Inhibition studies showed that non-labelled L-and D-methionine could both compete for binding with their labelled counterparts. These results suggest that PlpB is a novel, virulence-associated factor that is involved in the survival of P. multocida in chickens. Bioinformatics and structural studies of PlpB in comparison to the crystal structure of a related protein, Tp32, show that 8 of the 10 active site residues that interact with the methionine residue are identical, one is conserved, and the other one is a non-conserved substitution. In this work we report the vaccination results of five recombinant OM proteins of P. multocida. Out of the five proteins tested, the urea-solubilised recombinant PlpE showed protection in mice and chickens. Additional experiments in chickens and mice using insertionally inactivated mutants of all six genes showed that none of the encoded proteins played a significant role in the pathogenesis of P. multocida. Characterisation of PlpE by immunofluorescence microscopy showed it to be surface localised. This is the first report where a denatured recombinant protein, PlpE, has been shown to elicit protection against fowl cholera