The Capsule Is a Virulence Determinant in the Pathogenesis ofPasteurella multocidaM1404 (B:2)

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Abstract
Capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence inPasteurella multocida. We have previously identified and determined the nucleotide sequence of theP. multocidaM1404 (B:2) capsule biosynthetic locus (J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121–134, 2000). Thecaplocus 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 thetet(M) ΩcexAmutant 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 ofcexA. 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.