The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2)

Abstract

Capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence in Pasteurella multocida. We have previously identified and determined the nucleotide sequence of the P. multocida M1404 (B:2) capsule biosynthetic locus (J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). The cap locus consists of 15 genes, which can be grouped into three functional regions. Regions 1 and 3 contain genes proposed to encode proteins involved in capsule export, and region 2 contains genes proposed to encode proteins involved in polysaccharide biosynthesis. In order to construct a mutant impaired in capsule export, the final gene of region 1, cexA, was disrupted by insertion of a tetracycline resistance cassette by allelic replacement. The genotype of the tet(M) OmegacexA mutant was confirmed by Southern hybridization and PCR. The acapsular phenotype was confirmed by immunofluorescence, and the strain could be complemented and returned to capsule production by the presence of a cloned uninterrupted copy of cexA. Wild-type, mutant, and complemented strains were tested for virulence by intraperitoneal challenge of mice; the presence of the capsule was shown to be a crucial virulence determinant. Following intraperitoneal challenge of mice, the acapsular bacteria were removed efficiently from the blood, spleen, and liver, while wild-type bacteria multiplied rapidly. Acapsular bacteria were readily taken up by murine peritoneal macrophages, but wild-type bacteria were significantly resistant to phagocytosis. Both wild-type and acapsular bacteria were resistant to complement in bovine and murine serum.

FIG. 1

Schematic representation of construction of the capsule-deficient mutant of P. multocida M1404. (A) A 1,800-bp fragment containing all of cexB and part of cexA and cexC was amplified by PCR using the oligonucleotides BAP518 and BAP519 (designated by arrows labeled 518 and 519). Similarly, a 1,100-bp fragment containing part of cexA and downstream DNA was amplified by using the oligonucleotides BAP520 and BAP521 (designated by arrows labeled 520 and 521). These fragments were ligated to either end of the tet(M) gene to obtain the linear DNA (mutagenesis cassette) used for mutagenesis by allelic exchange. The line-and-ball characters indicate the location of a putative transcriptional terminator, proposed to constitute the end of the cap locus (4). (B) Integration of the fragment by double-crossover homologous recombination would give rise to Tet^r bacteria with inactive cexA. (C) The genotypes of three putative mutants were investigated by PCR. Genomic DNA was isolated from each mutant (lanes 1 to 3) and P. multocida M1404 (lane 4) and was used as a template for PCR with oligonucleotides BAP474 and BAP494. For comparison, the mutagenesis cassette DNA was also used as a template for PCR with the oligonucleotides BAP474 and BAP494 (lane 5). Lane 6 contains λ DNA digested with PstI. The sizes of the PCR products are indicated.

FIG. 2

Phenotypic characterization of P. multocida capsule expression. Photomicrographs of methanol-fixed P. multocida M1404 (A), PBA875 (B), and PBA1514 (C) visualized by immunofluorescence, with P. multocida serogroup B typing antiserum as the primary antibody and FITC-labeled anti-rabbit immunoglobulin as the secondary antibody.