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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.
Insights
The capsule of Pasteurella multocida is essential for its virulence, protecting bacteria from immune cells. Disrupting capsule export genes significantly reduces bacterial survival and virulence in mice.
Area of Science:
- Bacteriology
- Molecular Biology
- Immunology
Background:
- Bacterial capsules are critical virulence factors in many pathogens, including Pasteurella multocida.
- The capsule biosynthetic locus (cap locus) in P. multocida M1404 contains 15 genes involved in capsule production and export.
Purpose of the Study:
- To investigate the role of capsule export in Pasteurella multocida virulence.
- To construct and characterize a mutant with impaired capsule export.
Main Methods:
- Disruption of the cexA gene (region 1) in the P. multocida cap locus using tetracycline resistance cassette via allelic replacement.
- Genotypic confirmation using Southern hybridization and PCR.
- Phenotypic confirmation of acapsular state via immunofluorescence and complementation studies.
Main Results:
- A P. multocida mutant with disrupted cexA was successfully created and confirmed to be acapsular.
- The acapsular mutant showed significantly reduced virulence in a mouse model, with efficient clearance from blood and organs.
- Acapsular bacteria were susceptible to phagocytosis by macrophages, unlike wild-type bacteria which were resistant.
Conclusions:
- The P. multocida capsule is a crucial determinant of virulence, primarily by mediating resistance to phagocytosis.
- Capsule export, regulated by genes like cexA, is vital for bacterial survival and pathogenesis in vivo.