A molecular study of virulence factors of Bordetella species
A 1kb HinfI DNA fragment, containing a repeat DNA sequence element was isolated from a Bordetella pertussis BP348 cosmid gene bank. This repeat DNA sequence has subsequently been named IS148. The insertion sequence was demonstrated by have a high copy number only in B.pertissus and to be absent in Bordetella parapertussis and Bordetella bronchiseptica. Using a restriction fragment of IS148 labelled with radioactivity as a probe in DNA or whole cell dot blots between 10-100 cells could be detected or from 100pb-1ng DNA. Furthermore, a pair of oligonucleotide primers was synthesised corresponding to a central region of IS148 that could generate a 242bp fragment upon PCR amplification. The use of PCR amplification with specific primers allows the detection of 0.5pg of B.pertussis chromosomal DNA and as little as one B.pertussis cell. The prn genes encoding the outer membrane P.70 and P.68 pertactin from B.parapertussis and B.bronchiseptica have been cloned, sequenced and expressed in Escherichia coli. Analysis of the DNA sequences reveal that the genes have open reading frames capable of encoding proteins with molecular weights of 95,177 (P.95, B.parapertussis) and 93,996 (P.94, B.bronchiseptica). These precursors molecular are processed to form the P.70 and P.68 antigens on the surface of B.parapertussis and B.bronchiseptica respectively. Heterologous expression of the full-length gene encoding P.95 and P.94 in E.coli result in similar processing, with the P.70 and P.68 antigens targetted to the bacterial outer membrane. Comparison of P.95, P.94 and P.93, encoding homologous proteins from B.parapertussis, B.bronchiseptica and B.pertussis, shows a high degree (> 90%) of homology. The major differences between all three proteins occur in the number of repeat of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.