Rapid Method for Species-Specific Identification ofVibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (∼98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- andtoxR-specific probes in DNA dot blot assay. While theV. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus,V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxAgenes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

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Point mutations in the major outer membrane protein drive hypervirulence of a rapidly expanding clone of Campylobacter jejuni

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1605869113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10690-10695 ◽

Cited By ~ 35

Author(s):

Zuowei Wu ◽

Balamurugan Periaswamy ◽

Orhan Sahin ◽

Michael Yaeger ◽

Paul Plummer ◽

...

Keyword(s):

Sequence Analysis ◽

Membrane Protein ◽

Outer Membrane ◽

Campylobacter Jejuni ◽

Outer Membrane Protein ◽

Animal Health ◽

Major Outer Membrane Protein ◽

Whole Genome Sequence ◽

Bacterial Strains ◽

Zoonotic Pathogen

Infections due to clonal expansion of highly virulent bacterial strains are clear and present threats to human and animal health. Association of genetic changes with disease is now a routine, but identification of causative mutations that enable disease remains difficult. Campylobacter jejuni is an important zoonotic pathogen transmitted to humans mainly via the foodborne route. C. jejuni typically colonizes the gut, but a hypervirulent and rapidly expanding clone of C. jejuni recently emerged, which is able to translocate across the intestinal tract, causing systemic infection and abortion in pregnant animals. The genetic basis responsible for this hypervirulence is unknown. Here, we developed a strategy, termed “directed genome evolution,” by using hybridization between abortifacient and nonabortifacient strains followed by selection in an animal disease model and whole-genome sequence analysis. This strategy successfully identified SNPs in porA, encoding the major outer membrane protein, are responsible for the hypervirulence. Defined mutagenesis verified that these mutations were both necessary and sufficient for causing abortion. Furthermore, sequence analysis identified porA as the gene with the top genome-wide signal of adaptive evolution using Fu’s Fs, a population genetic metric for recent population size changes, which is consistent with the recent expansion of clone “sheep abortion.” These results identify a key virulence factor in Campylobacter and a potential target for the control of this zoonotic pathogen. Furthermore, this study provides general, unbiased experimental and computational approaches that are broadly applicable for efficient elucidation of disease-causing mutations in bacterial pathogens.

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Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

BMC Genomics ◽

10.1186/1471-2164-15-201 ◽

2014 ◽

Vol 15 (1) ◽

pp. 201 ◽

Cited By ~ 4

Author(s):

Kewei Li ◽

Wenpeng Gu ◽

Junrong Liang ◽

Yuchun Xiao ◽

Haiyan Qiu ◽

...

Keyword(s):

Membrane Protein ◽

Gene Polymorphism ◽

Outer Membrane ◽

Yersinia Enterocolitica ◽

Outer Membrane Protein ◽

Protein A ◽

Polymorphism Analysis ◽

Family Protein ◽

Outer Membrane Protein A

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The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes.

Infection and Immunity ◽

10.1128/iai.55.11.2570-2573.1987 ◽

1987 ◽

Vol 55 (11) ◽

pp. 2570-2573 ◽

Cited By ~ 12

Author(s):

Y X Zhang ◽

N G Watkins ◽

S Stewart ◽

H D Caldwell

Keyword(s):

Chlamydia Trachomatis ◽

Membrane Protein ◽

Molecular Mass ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Species Specific

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Faculty Opinions recommendation of Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1048393.502568 ◽

2006 ◽

Author(s):

Tracy Palmer

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein Substrates ◽

Assembly Factor ◽

Species Specific

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High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis.

Journal of Experimental Medicine ◽

10.1084/jem.167.3.817 ◽

1988 ◽

Vol 167 (3) ◽

pp. 817-831 ◽

Cited By ~ 78

Author(s):

R S Stephens ◽

E A Wagar ◽

G K Schoolnik

Keyword(s):

Amino Acid ◽

Chlamydia Trachomatis ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Antigenic Determinants ◽

Amino Acid Peptide ◽

Antigenic Domains ◽

Species Specific ◽

Specific Determinant

The principal surface protein antigen of Chlamydia trachomatis is the major outer membrane protein (MOMP). The MOMP is antigenically complex. Among the 15 serovars of C. trachomatis, mAbs define serovar-, subspecies-, and species-specific determinants on MOMP. The molecular basis of the antigenic diversity of these proteins is reflected in amino acid variable sequence domains. We have mapped the dominant topographic antigenic determinants of MOMP that are defined by mAbs. Using recombinant DNA approaches we have identified the linear distribution of two antigenic domains. One domain contains a serovar-specific determinant and the other contains subspecies- and species-specific determinants. These antigenic domains correspond to two amino acid sequence variable domains. Synthetic peptides were immunogenic and these resolved the serovar-specific determinant within a 14-amino acid peptide. The subspecies- and species-specific determinants were overlapping within a 16-amino acid peptide.

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Chlamydia pneumoniae Major Outer Membrane Protein Is a Surface-Exposed Antigen That Elicits Antibodies Primarily Directed against Conformation-Dependent Determinants

Infection and Immunity ◽

10.1128/iai.69.5.3082-3091.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3082-3091 ◽

Cited By ~ 45

Author(s):

Katerina Wolf ◽

Elizabeth Fischer ◽

David Mead ◽

Guangming Zhong ◽

Roseanna Peeling ◽

...

Keyword(s):

Monoclonal Antibody ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Chlamydia Pneumoniae ◽

Major Outer Membrane Protein ◽

Mass Spectrometry Analysis ◽

Antigenic Determinants ◽

Specific Monoclonal Antibody ◽

Species Specific

ABSTRACT The major outer membrane protein (MOMP) of Chlamydia trachomatis serovariants is known to be an immunodominant surface antigen. Moreover, it is known that the C. trachomatis MOMP elicits antibodies that recognize both linear and conformational antigenic determinants. In contrast, it has been reported that the MOMP of Chlamydia pneumoniae is not surface exposed and is immunorecessive. We hypothesized that the discrepancies betweenC. trachomatis and C. pneumoniae MOMP exposure on intact chlamydiae and immunogenic properties might be because the focus of the host's immune response is directed to conformational epitopes of the C. pneumoniae MOMP. We therefore conducted studies aimed at defining the surface exposure of MOMP and the conformational dominance of MOMP antibodies. We present here a description of C. pneumoniaespecies-specific monoclonal antibody (MAb), GZD1E8, which recognizes a conformational epitope on the surface of C. pneumoniae. This MAb is potent in the neutralization ofC. pneumoniae infectivity in vitro. Another previously described C. pneumoniaespecies-specific monoclonal antibody, RR-402, displayed very similar characteristics. However, the antigenic determinant recognized by RR-402 has yet to be identified. We show by immunoprecipitation ofC. pneumoniae with GZD1E8 and RR-402 MAbs and by mass spectrometry analysis of immunoprecipitated proteins that both antibodies GZD1E8 and RR-402 recognize the MOMP of C. pneumoniae and that this protein is localized on the surface of the organism. We also show that human sera fromC. pneumoniae-positive donors consistently recognize the MOMP by immunoprecipitation, indicating that the MOMP ofC. pneumoniae is an immunogenic protein. These findings have potential implications for both C. pneumoniae vaccine and diagnostic assay development.

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