RESUMO
Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.
Assuntos
Aderência Bacteriana , Campylobacter jejuni/metabolismo , Campylobacter jejuni/patogenicidade , Células Epiteliais/microbiologia , Flagelos/fisiologia , Flagelina/metabolismo , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Campylobacter jejuni/citologia , Campylobacter jejuni/genética , Linhagem Celular , Citoplasma/microbiologia , Células Epiteliais/metabolismo , Flagelos/metabolismo , Flagelina/genética , Deleção de Genes , Genes Bacterianos , Humanos , Movimento , Mutagênese , Transporte Proteico , Homologia de Sequência de AminoácidosRESUMO
Currently, the detection and identification of Campylobacter and Arcobacter species remains arduous, largely due to cross-species phenotypic similarities and a relatively narrow spectrum of biochemical reactivity. We have developed a PCR-hybridization strategy, wherein degenerate primers are used to amplify glyA fragments from samples, which are then subjected to species-specific oligodeoxyribonucleotide probe hybridizations, to identify and distinguish between Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, Arcobacter butzleri, and an A. butzleri-like species. Evaluation of this strategy with genomic DNA from different type strains suggests that this approach is both specific and sensitive and thus may be applicable in a diagnostic assay to identify and differentiate these highly related species.
Assuntos
Arcobacter/classificação , Campylobacter/classificação , Glicina Hidroximetiltransferase/genética , Reação em Cadeia da Polimerase/métodos , Arcobacter/enzimologia , Sequência de Bases , Southern Blotting , Campylobacter/enzimologia , Infecções por Campylobacter/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The taxonomic affiliation of Campylobacter hyoilei was reevaluated by examining a variety of phenotypic and genotypic criteria. Whole-cell protein electrophoresis and a comparison of 66 phenotypic characters revealed that reference strains of C. hyoilei were indistinguishable from Campylobacter coli strains. These data were confirmed by a DNA-DNA hybridization level of 67% between the type strains of the two species. Several species-specific assays based on PCR amplification or probe hybridization further substantiated that C. coli strains and C. hyoilei strains are indistinguishable. It is therefore concluded that C. hyoilei and C. coli represent the same species and that the former name should be regarded as a junior synonym of the latter name.
Assuntos
Campylobacter coli/classificação , Campylobacter coli/genética , Genes Bacterianos/genética , Proteínas de Bactérias/análise , Campylobacter coli/metabolismo , Eletroforese em Gel de Poliacrilamida , GTP Fosfo-Hidrolases/genética , Hibridização de Ácido Nucleico , Fenótipo , Filogenia , Reação em Cadeia da PolimeraseRESUMO
Despite increasing recognition of the importance of Campylobacter upsaliensis in human disease little is known about either the virulence properties or genetics of this enteric pathogen. The complete coding sequence of a C. upsaliensis gene has yet to be published. We have cloned and sequenced the complete iron-uptake regulatory (fur) gene from the type strain of this species. The C. upsaliensis fur homolog was isolated from a genomic library of C. upsaliensis ATCC 43954 constructed in phage lambdaGEM-11. The open reading frame identified encodes a polypeptide consisting of 156 amino acids. The 5'-flanking region of the C. upsaliensis fur gene contains 3 putative Fur-binding sequences and two catabolite activator-binding sequences indicating the potential for autogenous and cAMP-mediated regulation, respectively. Primer extension analysis identified a single transcription start site 262 nt upstream from the AUG initiation codon. Sequence analysis indicates that the Fur protein of C. upsaliensis is highly homologous (87% amino acid identity) to Campylobacter jejuni Fur. Furthermore, the arrangement of the lysS and glyA genes downstream of fur is precisely conserved in both C. upsaliensis ATCC 43954 and C. jejuni TGH9011. Using the polymerase chain reaction close linkage of fur with lysS and glyA was also observed among multiple isolates of C. upsaliensis, C. jejuni and C. coli suggesting a possible functional relevance for this conserved genetic arrangement in campylobacteria.
