Genomics of Bordetella pertussis toxins

Bordetella pertussis, the etiologic agent of whooping cough, produces numer ous toxins including pertussis toxin (PTX), adenylate cyclase toxin (AC), d ermonecrotic toxin (DNT) and tracheal cytotoxin (TCT). PTX is composed of f ive different subunits organised in a typical A-B type structure of which t he A part possesses an enzymatic ADP-ribosyltransferase activity and the B moiety expresses receptor-binding activity. The secretion of this toxin req uires 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 seq uences. Comparison of these regions between three species of Bordetella (B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica) reveale d differences in the upstream region. Analysis of two strains of B. bronchi septica 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 homologou s with putative proteins from several other Gram-negative organisms. Detail ed analysis of the genome of B. pertussis which is currently sequenced at t he Sanger Centre revealed the presence of 90 genes coding for proteins homo logous to BugT, which qualifies the bug gene family as the most populated o ne of Bordetella. These bug genes are located in Various genetic environmen ts, 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 periodi city of predicted secondary structure elements, but have also a high variab ility in their amino acid composition reflected in their wide range of isoe lectric points. The function of these genes which is currently unknown is u nder investigation. To characterise the expression and regulation of these genes, as well as of novel putative B. pertussis Virulence factors, we desi gned 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 ge nes are expressed.