Article

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73

Department of Avian Medicine, College of Veterinary Medicine, The University of Georgia, Athens 30602, USA.
Journal of Bacteriology (Impact Factor: 2.81). 01/1998; 179(24):7856-64.
Source: PubMed

ABSTRACT

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.

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Available from: Manjeet Bains, Sep 02, 2014
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    • "An intervention study was used with five treatment groups; there were four immunized groups and one nonimmunized group (Table 1). The treatments included group 1, immunized with rOmpH purified using electroelution, group 2, immunized with rOmpH purified using a denatured condition of affinity chromatography [1], group 3, immunized with native OmpH purified using electroelution [8], group 4, immunized with incomplete Freund's adjuvant, and group 5, a nonimmunized group, respectively. Each of the five treatment groups was divided into two subgroups, one challenged with P. multocida serovar A:1 and the other with serovar A:3. "
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    ABSTRACT: Recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 can be purified using affinity chromatography but this adversely affects its immunogenicity. The current study presents the results from an intervention study comparing the immunogenicity of rOmpH purified using electroelution with rOmpH purified using affinity chromatography and native OmpH purified using electroelution and a nonimmunized control group. Chickens immunized with rOmpH purified using electroelution produced the highest ELISA antibody levels against P. multocida strains. Chickens in each of the 5 treatment groups were split into two subgroups for challenge with two different P. multocida strains. The average number of adhesions to CEF cells was statistically significantly lower in sera from chickens immunized with rOmpH or native OmpH purified using electroelution than in those of the three other treatment groups. The survival amongst chickens immunized with rOmpH or native OmpH purified using electroelution indicated high levels of protection. In contrast, survival probability was zero or low in the groups immunized with rOmpH purified using affinity chromatography and in the nonimmunized group. These findings show that the rOmpH purified using electroelution retains its immunogenicity and stimulates high levels of protection in chickens against P. multocida infection.
    Full-text · Article · Jan 2016 · Veterinary Medicine International
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    • "The OmpC protein sequence shows 40% similarity to the pore-forming OmpH protein from Pasterella multocida (Chevalier et al., 1993). The OmpH protein is a conserved OM porin with immunogenic potential (Luo et al., 1997). The identification of this protein as a constant entity of G. anatis OMVs may suggest a potential of the OMVs as a vaccine candidate, however, further studies are needed to elucidate this. "
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    ABSTRACT: The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg-production worldwide. Increased knowledge of the pathogenesis and virulence factors is important to better understand and prevent the negative effects of G. anatis. To this end outer membrane vesicles (OMVs) are natural secretion products of Gram-negative bacteria, displaying an enormous functional diversity and promising results as vaccine candidates. This is the first study to report that G. anatis secretes OMVs during in vitro growth. By use of transmission electron microscopy (TEM) and SDS-PAGE, we showed that changes in in vitro growth conditions, including incubation time, media composition and temperature, affected the OMV production and protein composition. A large protein band was increased in its concentration after prolonged growth. Analysis by LC-MS/MS indicated that the band contained two proteins; the 320.1kDa FHA precursor, FhaB, and a 407.8kDa protein containing a von Willebrand factor type A (vWA) domain. Additional two major outer-membrane (OM) proteins could be identified in all samples; the OmpH-homolog, OmpC, and OmpA. To understand the OMV formation better, a tolR deletion mutation (ΔtolR) was generated in G. anatis. This resulted in a constantly high and growth-phase independent production of OMVs, suggesting that depletion of peptidoglycan linkages plays a role in the OMV formation in G. anatis. In conclusion, our results show that G. anatis produce OMVs in vitro and the OMV protein profile suggests that the production is an important and well-regulated ability employed by the bacteria, which may be used for vaccine production purposes.
    Full-text · Article · Sep 2013 · Veterinary Microbiology
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    • "Wu et al. (2007) reported that rPlpE from P. multocida serotype A:1 causing fowl cholera conferred 63–100% protection in mice and chickens against challenge with serotypes A:1, A:3 and A:4. Luo et al. (1997) vaccinated chickens with native and recombinant OmpH from P. multocida serotype A:1 and reported 100% and 18% protection, respectively. However, Sthitmatee et al. (2008) showed that both native and recombinant OmpH from serotype A:1 and its identical protein, Cp39 from serotype A:3, conferred 60–100% protection in chickens against challenge with serotypes A:1 and A:3. "
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    ABSTRACT: Pasteurella multocida serotype A:3 is a Gram-negative bacterial pathogen, one of the causative agents of shipping fever of cattle. In this study, outer membrane protein H (ompH) and Pasteurella lipoprotein E (plpE) genes were cloned and plpEC-ompH fusion was constructed and expressed in Escherichia coli. Recombinant PlpE, OmpH and PlpEC-OmpH fusion proteins were purified and formulated with oil-based and oil-based CpG ODN adjuvants. Antibody responses in mice vaccinated with recombinant PlpE and PlpEC-OmpH proteins formulated with both adjuvants were significantly (p<0.05) increased. However, a significant (p<0.05) increment in serum IFN-γ level was only observed upon immunization with oil-based CpG formulations. Protectivity of the vaccines were evaluated via intraperitoneal challenge of mice with 10 LD(50) of P. multocida A:3. The recombinant proteins PlpE and PlpEC-OmpH fusion conferred 100% protection when formulated with oil-based CpG ODN while the protectivity was found to be 80% and 60%, respectively when only oil-based adjuvant was used in respective formulations. These findings indicated that the recombinant PlpE or PlpEC-OmpH fusion proteins formulated with oil-based CpG ODN adjuvant are possible acellular vaccine candidates against shipping fever.
    Full-text · Article · Jun 2012 · Research in Veterinary Science
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