Kumar Rajakumar

Candidate vaccine antigens and genes in Pasteurella multocida

Pasteurella multocida is the causative agent of fowl cholera and other diseases of production animals. Isolates are classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens. Strains causing fowl cholera are most frequently designated A:1, A:3 or A:4. Whole cell bacterins can provide some degree of protection, but only against the homologous LPS serotype. There is good evidence that cross-protective antigens are expressed only under in vivo conditions. Empirically derived, live, attenuated vaccines can protect against heterologous serotypes, but because the basis for attenuation is undefined, reversion to virulence is not uncommon. Work in our laboratory is aimed at using a variety of approaches to identify potential protective antigens or virulence genes to be used as candidates for attenuating mutations or as the basis for vaccine antigen delivery systems. The gene encoding an outer membrane protein, Oma87, which is a homologue of the D15 protective antigen of Haemophilus influenzae, was cloned and sequenced. Rabbit antiserum prepared against recombinant Oma87 could passively protect mice against infection. Type 4 fimbriae form the basis of vaccines against ovine footrot and bovine keratoconjunctivitis. We have identified type 4 fimbriae on the surface of P. multocida, purified the fimbrial subunit protein, PtfA, and determined its N-terminal amino acid sequence. Subsequent cloning of the ptfA gene and its inactivation will now be used to assess the importance of type 4 fimbriae in virulence. There has long been anecdotal evidence for the importance of capsule in virulence, but unequivocal genetic evidence for such a role is lacking. We have cloned and characterised the capsule biosynthetic locus in P. multocida A:1 and identified four bex genes involved in capsule transport and genes encoding enzymes involved in the biosynthesis and transfer of the N-acetyl glucosamine and glucuronic acid components of the capsule. It has been suggested that the low concentration of available iron in vivo acts as an environmental cue for expression of cross-protective antigens. Accordingly, we have cloned and characterised the gene encoding transferrin binding protein, Tbpl, so that its role in immunity and virulence can be investigated. Although P. multocida is not normally considered haemolytic, we have observed haemolysis under anaerobic conditions. Standard library construction and screening resulted in the identification of the mesA gene which encodes an esterase enzyme resulting in a haemolytic phenotype under anaerobic conditions. Virulence studies with mesA- mutants were performed to assess its role in pathogenesis. Using a promoterless phoA gene vector system, the cloning of proteins homologous to known surface proteins of other species as well as proteins unique to P. multocida, allowing their potential as vaccine components to be assessed.

Regulated site‐specific recombination of the she pathogenicity island of Shigella flexneri

Summary The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3′ terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase‐mediated excision occurs via recombination between a 22 bp sequence at the 3′ terminus of pheV and an imperfect direct repeat at the pheV‐distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase‐mediated integration into the chromosome. The circle junction consists of the pheV‐proximal and pheV‐distal boundaries of the PAI converging on a sequence identical to 22 bp at the 3′ terminus of pheV. The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri, independently of recA. We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase‐dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2.

Distribution and structural variation of the she pathogenicity island in enteric bacterial pathogens

Shigella flexneri serotype 2a carries a chromosomal pathogenicity island (PAI), termed the she PAI, that has been implicated in the pathogenesis of diarrhoeal disease. The complete nucleotide sequence and genetic organisation of the she PAI of S. flexneri 2a strain YSH6000T was determined recently. In the current study the distribution and structure of the she PAI was investigated by PCR and Southern analysis in 65 isolates of enteric pathogens including Shigella spp., enterohaemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Yersinia enterocolitica and Salmonella enterica serovar Typhimurium. The study showed that the she PAI has undergone a variety of structural changes, defined by the presence or absence of specific marker genes in the PAI. The she PAI or structural variants of this element were found in all species of Shigella as well as in EIEC, EHEC and EPEC. No evidence of the PAI was found in Y. enterocolitica or Sal. Typhimurium. The structural form of the she PAI that exists in strain YSH6000T was present in all strains of S. flexneri serotype 2a and in some strains of S. flexneri serotypes 2b and 3c. Variants of the PAI that were missing one or more marker regions were found in all species of Shigella and in pathogenic strains of E. coli. In all strains, the PAIs have inserted into either pheV or a phe tRNA gene in another location on the chromosome. It was concluded that the she PAI is one of several closely related genetic elements that have disseminated throughout Shigella and pathogenic strains of E. coli and diverged into distinct stuctural forms.

Regulated site-specific recombination of the she pathogenicity island of Shigella flexneri.

Summary The she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3′ terminus of the pheV tRNA gene is mediated by an integrase gene (int) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase‐mediated excision occurs via recombination between a 22 bp sequence at the 3′ terminus of pheV and an imperfect direct repeat at the pheV‐distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase‐mediated integration into the chromosome. The circle junction consists of the pheV‐proximal and pheV‐distal boundaries of the PAI converging on a sequence identical to 22 bp at the 3′ terminus of pheV. The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri, independently of recA. We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase‐dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2.

A spontaneous 99-kb chromosomal deletion results in multi-antibiotic susceptibility and an attenuation of contact haemolysis in Shigella flexneri 2a

A Tn5-generated mutant (strain S2430) of Shigella flexneri 2a (strain YSH6000) exhibited attenuated virulence and, in addition to the Tn5 insertion in the SalI K fragment of its virulence plasmid, had a 99-kb deletion within its chromosome. Unlike its wild-type parent, strain S2430 was susceptible to ampicillin, streptomycin, tetracycline and chloramphenicol. An independent multi-antibiotic susceptible variant of strain YSH6000 had a similar deletion. Southern blot analysis of pulsed field electrophoresis gels enabled the sizing of this deletion and its mapping to a region of the chromosome on NotI fragment D bounded by the S. flexneri homologues of ompA and pyrC. Hybridisation experiments with a probe specific to the multi-antibiotic resistance region indicated that this large deletion was responsible for antibiotic susceptibility. Both strain S2430 and a derivative of the antibiotic-susceptible variant, with a Tn5 insertion in its SalI K fragment, exhibited an equal reduction in contact haemolysis compared with the Tn5-bearing derivative of strain YSH6000. However, strain S2430 alone clearly displayed delayed plaque forming ability in LLC-MK2 monolayers, suggesting that the two examples of this deletion may not be identical.

Evolutionary perspective on a composite Shigella flexneri 2a virulence plasmid-borne locus comprising three distinct genetic elements

Nucleotide sequence analysis of a Shigella flexneri 2a virulence plasmid-borne locus revealed that it comprised three distinct genetic elements: a stretch of colicin 1a/1b-linked sequence, a truncated IS911 element, and a third element containing two ORFs that shared a high level of similarity to a Salmonella-specific chromosomal sequence. Examination of other known IS911-like sequences showed that these sequences also were frequently associated with other accessory elements and appeared to be prone to partial deletion events. Analysis of the data led to a model of the evolution of this unusual composite locus.