Role of lipopolysaccharide of actinobacillus pleuropneumoniae in biofilm formation and pathogenesis

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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 etiological agent of porcine pleuropneumonia, is our model. We have generated a collection of lipopolysaccharide (LPS) mutants of App using transposon mutagenesis. We have shown that App LPS is involved in colonization of porcine respiratory tract and that deletion of specific residues in the LPS outer core results in the attenuation of the virulence of App. We hypothesize that LPS are involved in biofilm formation in App and that biofilm formation is a complex and finely regulated process which plays an important role in pathogenesis. The short-term objectives of this research program are: (1) to characterize the relation between LPS and biofilm formation. We will first characterize the collection of App LPS mutants we have generated and determine their ability to form biofilms. We will use a standard static biofilm assay in microtiter plates as well as a drip-flow cell which is more representative of the air-liquid interface found in the lungs. We will use phage display technology to select peptides with a strong affinity for purified LPS molecules. We will then quantify the affinity of these peptides using surface plasmon resonance and determine whether these peptides can interfere with biofilm formation or with other biological activities of LPS. (2) to use genomic (transposon mutagenesis) and transcriptomic (DNA microarray) approaches to identify additional genes involved in App biofilm formation. A better understanding of surface polysaccharides of App and of the genes expressed during biofilm formation will lead to the development of effective infection-control strategies such as novel molecules that can block the activity of important virulence factors or inhibit biofilm formation of App and, potentially, other pathogens of farm animals.