J HAREL, Université de Montréal, Faculté de Médecine Vétérinaire F DAIGLE, Université de Montréal
Title
Gene Knock Outs Strategy to Study the Role of the Pst System in Pathogenic Escherichia coli Virulence
Abstract Text
Escherichia coli 0115:K”V165”:F165 strains have been associated with septicemia in calves and piglets and they are resistant to the bactericidal effects of serum. We had previously obtained an avirulent, serum-sensitive mutant in the pst operon of the pathogenic strain by transposon mutagenesis. The pst-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport system (Pst). The Pho regulon is controlled by a two-component regulatory system, PhoB/PhoR, which responds to phosphate concentration. When grown in conditions of phosphate starvation, the E. coli Pho regulon is induced whereas its expression is inhibited when phosphorus is in excess. Constitutive expression of the Pho regulon was observed in Pst mutants. Pst mutant demonstrated an increased sensitivity to the bactericidal effects of serum and to acid shock. The hypothesis is that the bacterial cell surface of the mutants is modified. Polar (DpstCAB::kan) and non-polar (pstC::kan) mutations in the pst operon were created by allelic exchange. In vitro acid shock tests were carried out on stationary phase cultures without habituation. The mutants were tested by reaction with K”V165” antiserum and quantification of their capsular carbohydrate content. The LPS as well as the lipid A of the mutant strains were analyzed by SDS-PAGE and Tricine SDS-PAGE, respectively, and by Western Blot analysis. The capacity to resist acid shock was impaired in pst mutants in contrast to wild-type. This effect was a function of time. Complementation of the mutants restored this property. pst mutants produced less K”V165” capsular antigen than the wild type strain and the complement. However, no major differences were observed in LPS and lipid A profiles. Mutations in Pst affect the ability of the pathogenic strain to survive adverse environmental conditions such as acidic conditions and it increases the sensitivity to the bactericidal effects of serum. Although pst mutants produced less K”V165” capsular antigen, it is not known how the pst mutations influence the modifications of the bacterial surface.
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