RESUMO
Investigation of the hemolytic phenotype under anaerobic growth conditions of an avian Pasteurella multocida strain, PBA100, resulted in the identification and characterisation of a gene encoding an esterase enzyme, mesA, that conferred a hemolytic phenotype in Escherichia coli under anaerobic conditions. MesA appeared to be expressed and functional under anaerobic and aerobic conditions in both E. coli and P. multocida. A P. multocida mesA mutant was generated which resulted in the loss of acetyl esterase activity under anaerobic conditions. However, this mutation did not cause any attenuation of virulence for mice nor a detectable change to the anaerobic hemolytic phenotype of P. multocida. In E. coli MesA appeared to cause hemolysis indirectly by the induction of the latent E. coli K-12 cytolysin, sheA.
Assuntos
Proteínas de Bactérias/genética , Esterases , Genes Bacterianos , Pasteurella multocida/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bovinos , Clonagem Molecular , Escherichia coli/genética , Hemólise/genética , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Pasteurella multocida/enzimologia , Pasteurella multocida/patogenicidade , Fenótipo , Alinhamento de Sequência , Ovinos , Suínos , VirulênciaRESUMO
Colonisation of host tissue by Gram- negative bacteria is facilitated by various adhesins, one of which is type 4 fimbriae (pili). These structures have been associated with pathogenesis in several bacterial species, and have been shown to mediate colonisation of epithelial surfaces. Recently, type 4 fimbriae were identified and characterised from P. multocida strains A, B and D. The type 4 fimbrial subunit protein (PtfA) was identified as an 18-kDa protein which was isolated from whole membrane fractions. We report here the isolation and characterisation of the gene (ptfA) encoding the PtfA protein from P. multocida VP161 (serotype A:1). Part of the gene was cloned on a 2-kb genomic DNA fragment. The complete ptfA gene was obtained using inverse PCR. The gene and its flanking regions were characterised, and the deduced PtfA amino acid sequence was compared to type 4 subunit protein sequences from other bacterial species. The ptfA gene was amplified and sequenced from several P. multocida strains. Comparison of these sequences revealed variation within the type 4 subunit gene of P. multocida.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/genética , Pasteurella multocida/genética , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Southern Blotting , DNA Bacteriano/química , Proteínas de Ligação a DNA/genética , Dados de Sequência MolecularRESUMO
Haemolysins are membrane-damaging agents which have been described as bacterial virulence factors due to their ability to lyse erythrocytes and other host cells, and therefore inducing a greater inflammatory response (Elliott et al., 1998). Pasteurella multocida was found to be haemolytic under anaerobic conditions. In this study, we cloned and characterised a P. multocida gene, designated ahpA, which conferred a haemolytic phenotype on Escherichia coli when incubated under anaerobic conditions. A deletion was introduced into the ahpA open reading frame which abolished the haemolytic phenotype. The clone containing ahpA showed erythrocyte specificity, causing haemolysis of bovine and equine erythrocytes, and demonstrated weak haemolysis on ovine erythrocytes. Upon further investigation, AhpA was found to affect the expression of the E. coli K-12 latent haemolysin, SheA, under anaerobic conditions.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Pasteurella multocida/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Hemólise , Dados de Sequência Molecular , Fenótipo , Análise de SequênciaRESUMO
A physical and genetic map of the Pasteurella multocida A:1 genome was generated by using the restriction enzymes ApaI, CeuI, and NotI. The positions of 23 restriction sites and 32 genes, including 5 rrn operons, were localized on the 2.35-Mbp single circular chromosome. This report presents the first genetic and physical map for this genus.
Assuntos
Mapeamento Cromossômico , Cromossomos Bacterianos , Pasteurella multocida/genética , Mapeamento por Restrição , Genes Bacterianos , Genoma Bacteriano , ÓperonAssuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Ferro/metabolismo , Infecções por Pasteurella/veterinária , Pasteurella multocida/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting/veterinária , Centrifugação com Gradiente de Concentração/veterinária , Galinhas , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pasteurella/imunologiaRESUMO
The presence of fimbriae on Pasteurella multocida has been reported, but there have been no prior studies aimed at conclusively characterizing these structures. We now report on the identification and characterization of type 4 fimbriae on serogroup A, B, and D strains of P. multocida. Under microaerophilic conditions P. multocida showed an increased expression of the fimbriae, which were observed to form bundles. Fimbriae purified by high-performance reverse-phase liquid chromatography constituted a single 18-kDa subunit, the first 21 amino acids of which shared very high similarity with the N-terminal amino acid sequence of other type 4 fimbrial subunits. Antiserum against the P. multocida 18-kDa protein immunostained the type 4 fimbrial subunit of Moraxella bovis and Dichelobacter nodosus. Based on these observations we conclude that P. multocida possesses type 4 fimbriae and have designated the P. multocida fimbrial subunit PtfA.
Assuntos
Proteínas de Fímbrias , Fímbrias Bacterianas/química , Pasteurella multocida/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Reações Cruzadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/imunologia , Fímbrias Bacterianas/classificação , Dados de Sequência Molecular , Pasteurella multocida/classificação , Pasteurella multocida/imunologia , Análise de Sequência , Homologia de Sequência , SorotipagemRESUMO
Membrane proteins of Pasteurella multocida have been shown previously to elicit protective immunity. We have identified an 87-kDa outer membrane antigen, Oma87, which is present in all 16 serotypes of P. multocida. The gene encoding this protein was cloned and sequenced and found to have significant similarity to the D15 protective surface antigen of Haemophilus influenzae. Oma87 was localized to the outer membrane of the cell, and proteinase K treatment suggested that the protein is surface exposed. Native and recombinant Oma87 were strongly immunostained by convalescent-phase antiserum, indicating that the protein is expressed in vivo. Specific Oma87 antiserum protected mice against homologous, lethal P. multocida challenge. These results suggest that Oma87 is a protective outer membrane antigen of P. multocida.