Dataset Information

Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella.

ABSTRACT: Salmonella isolates have traditionally been classified by serotyping, the serologic identification of two surface antigens, O-polysaccharide and flagellin protein. Serotyping has been of great value in understanding the epidemiology of Salmonella and investigating disease outbreaks; however, production and quality control of the hundreds of antisera required for serotyping is difficult and time-consuming. To circumvent the problems associated with antiserum production, we began the development of a system for determination of serotype in Salmonella based on DNA markers. To identify flagellar antigen-specific sequences, we sequenced 280 alleles of the three genes that are known to encode flagellin in Salmonella, fliC, fljB, and flpA, representing 67 flagellar antigen types. Analysis of the data indicated that the sequences from fliC, fljB, and flpA clustered by the antigen(s) they encode not by locus. The sequences grouped into four clusters based on their conserved regions. Three of the four clusters included multiple flagellar antigen types and were designated the G complex, the Z4 complex, and the alpha cluster. The fourth cluster contained a single antigen type, H:z(29). The amino acid sequences of the conserved regions within each cluster have greater than 95% amino acid identity, whereas the conserved regions differ substantially between clusters (75 to 85% identity). Substantial sequence heterogeneity existed between alleles encoding different flagellar antigens while alleles encoding the same flagellar antigen were homologous, suggesting that flagellin genes may be useful targets for the molecular determination of flagellar antigen type.

SUBMITTER: McQuiston JR

PROVIDER: S-EPMC404605 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella.

McQuiston J R JR Parrenas R R Ortiz-Rivera M M Gheesling L L Brenner F F Fields P I PI

Journal of clinical microbiology 20040501 5

Salmonella isolates have traditionally been classified by serotyping, the serologic identification of two surface antigens, O-polysaccharide and flagellin protein. Serotyping has been of great value in understanding the epidemiology of Salmonella and investigating disease outbreaks; however, production and quality control of the hundreds of antisera required for serotyping is difficult and time-consuming. To circumvent the problems associated with antiserum production, we began the development o ...[more]

PMID: 15131150

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Project description:Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170,018, 5933, 5939, and PAK) were compared, and that of 170,018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170,018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170,018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170,018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.

Project description:Clostridium difficile is the main agent responsible for hospital acquired antibiotic associated diarrhoea. In recent years, epidemic strains have emerged causing more severe infections. Whilst C. difficile has two major virulence factors, toxins TcdA and TcdB, it is generally accepted that other virulence components of the bacterium contribute to disease. Previously, it has been suggested that flagella expression from pathogenic bacteria might be implicated in virulence. In a recent study, we observed an increased mortality in a gnotobiotic mouse model when animals were colonized with an isogenic fliC mutant constructed in the PCR-ribotype 027 (B1/NAP1) strain R20291, while animals survived when colonized by the parental strain or after colonization by other high-toxin-producing C. difficile strains. To understand the reasons for this increased virulence, we compared the global gene expression profiles between the fliC-R20291 mutant and its parental strain using an in vitro and in vivo transcriptomic approach. The latter made use of the gnotobiotic mouse model. Interestingly, in the fliC mutant, we observed considerable up-regulation of genes involved in mobility, membrane transport systems (PTS, ABC transporters), carbon metabolism, known virulence factors and sporulation. A smaller but significant up-regulation of genes involved in cell growth, fermentation, metabolism, stress and antibiotic resistance was also apparent. All of these genes may be associated with the increased virulence of the fliC-R20921 mutant. We confirmed that the fliC mutation is solely responsible for the observed changes in gene expression in the mutant strain since expression profiles were restored to that of the wild-type strain in the fliC-complemented strain. Thus, the absence of FliC is directly or indirectly involved in the high mortality observed in the fliC mutant infected animals. Therefore, we provide the first evidence that when the major structural component of the flagellum is neutralized, deregulation of gene expression can occur during infection.

Dataset Information

Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella.

Publications

Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella.

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