Genomics of Bordetella pertussis toxins.

Bordetella pertussis, the etiologic agent of whooping cough, produces numerous toxins including pertussis toxin (PTX), adenylate cyclase toxin (AC), dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT). PTX is composed of five different subunits organised in a typical A-B type structure of which the A part possesses an enzymatic ADP-ribosyltransferase activity and the B moiety expresses receptor-binding activity. The secretion of this toxin requires nine other genes (ptl) organised in an operon together with the five structural genes of PTX. To further characterise the genetic locus of this major virulence factor, we analysed the ptx/ptl upstream and downstream sequences. Comparison of these regions between three species of Bordetella (B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica) revealed differences in the upstream region. Analysis of two strains of B. bronchiseptica naturally lacking the ptx genes showed that only the ptx/ptl genes were deleted in these strains, and that the upstream and downstream regions were conserved. Upstream of the PTX structural genes and the promoter, an open reading frame (bugT) was identified, the product of which is homologous with putative proteins from several other Gram-negative organisms. Detailed analysis of the genome of B. pertussis which is currently sequenced at the Sanger Centre revealed the presence of 90 genes coding for proteins homologous to BugT, which qualifies the bug gene family as the most populated one of Bordetella. These bug genes are located in various genetic environments, including the proximities of genes coding for other toxins, such as DNT and AC. The Bug proteins are highly conserved in terms of size and periodicity of predicted secondary structure elements, but have also a high variability in their amino acid composition reflected in their wide range of isoelectric points. The function of these genes which is currently unknown is under investigation. To characterise the expression and regulation of these genes, as well as of novel putative B. pertussis virulence factors, we designed a transcriptional fusion vector to be inserted in precise locations of the B. pertussis chromosome by homologous recombination. The reporter gene present in this vector allowed us to show that at least some of the bug genes are expressed.