Interaction of actinobacillus pleuropneumoniae with host epithelial cells

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The long-term objective of this research program is to gain a better understanding of the virulence factors involved in the Gram-negative bacterial pathogens colonization of the host's respiratory tract. Actinobacillus pleuropneumoniae (App), the agent of porcine pleuropneumonia, is our model. Our hypothesis is that App is able to express multiple adhesins and  that these adhesins are involved, at one time or another, during the colonization and infection process. We have shown that lipopolysaccharide (LPS) is one of the adhesins of App involved in adherence to porcine respiratory tract cells. In order to identify which part of the LPS molecule (core oligosaccharide or O-antigen) is involved in binding to these cells, we have generated LPS mutants of App serotype 1 using mini-Tn10 transposon mutagenesis. Our data suggest that deletion of specific residues in the outer LPS core affects colonization and results in the attenuation of the virulence of App serotype 1 and that an intact core-lipid A region is required for optimal protection of App against host cationic antimicrobial peptides. The short-term objectives of this research program are: (1) to further characterize the biological properties of the LPS mutants we have generated in our laboratory. We will compare the parental strain and LPS mutants' capacity to stimulate cytokines production by lung epithelial cells; (2) to develop an in vitro adherence assay using porcine lung (SJPL) and trachea (NPTr) epithelial cell lines; (3) to determine the transcriptome profiling of App in contact with host cells using DNA microarrays which will enable us to get for the first time a global view on App genes that are specifically expressed after interaction with host cells; (4) to generate novel mutants in putative adhesins by allelic exchange (on selected genes) or by random transposon mutagenesis. A better understanding of the adhesin-receptor systems of App and of the genes expressed upon contact with host epithelial cells will lead to the development of more efficient strategies to control infection.