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Molecular and immunological characterization of Pasteurella multocida serotype A:3 OmpA: evidence of its role in P.
S M Dabo1, A W Confer, R A Quijano-Blas
1Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078-2007, USA. mdvpath@okstate.edu
Abstract:
Pasteurella multocida OmpA-like gene (PmOmpA) was cloned and characterized. The mature protein had a molecular mass of 35,075 Da and significant similarity with Escherichia coli (E. coli) OmpA proteins. Membrane topology analyses predict that like E. coli OmpA, the N-terminal half of PmOmpA exists as an eight-stranded transmembrane antiparallel beta-barrel that displays four variable hydrophilic and surface-exposed regions with predicted antigenic peaks that may be involved in serum resistance or adherence. In addition, the Ala-Pro repeat region between the N-terminal beta-barrel and C-terminal periplasmic domains is completely missing in PmOmpA. PmOmpA was expressed in E. coli and immunoblots analysis revealed that the recombinant PmOmpA was immunogenic, and expressed in vivo. The binding of PmOmpA to biotinylated Madin Darby bovine kidney (MDBK) cells surface proteins, fibronectin and heparin was demonstrated. Furthermore, PmOmpA binds MDBK monolayers and pre-treatment of P. multocida whole cells with anti-PmOmpA significantly reduced adherence to fibronectin. Ligand blot analysis revealed that fibronectin binds to the native and heat modified forms of PmOmpA when heated at 37 and 100 degrees C, respectively. Collectively these data indicate that PmOmpA may be involved in P. multocida 232 adherence to host cells via heparin and/or fibronectin bridging.
Insights
The Pasteurella multocida OmpA-like gene (PmOmpA) protein may help the bacteria stick to host cells. This protein binds to fibronectin and heparin, potentially aiding in adherence.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Pasteurella multocida is a significant veterinary pathogen.
- Outer membrane proteins (OMPs) play crucial roles in bacterial interactions with host cells.
- The OmpA-like protein from P. multocida (PmOmpA) has not been fully characterized.
Purpose of the Study:
- To clone and characterize the Pasteurella multocida OmpA-like gene (PmOmpA).
- To investigate the role of PmOmpA in bacterial adherence to host cells.
- To determine the binding properties of PmOmpA to host cell surface components.
Main Methods:
- Gene cloning and protein expression in Escherichia coli.
- Bioinformatic analysis of protein structure and topology.
- Immunoblotting and immunogenicity assays.
- Cell surface binding assays with Madin Darby bovine kidney (MDBK) cells, fibronectin, and heparin.
- Ligand blot analysis to assess protein binding under different conditions.
Main Results:
- PmOmpA shares structural similarities with E. coli OmpA, featuring a beta-barrel structure.
- Recombinant PmOmpA was immunogenic and expressed in vivo.
- PmOmpA directly binds to MDBK cell surface proteins, fibronectin, and heparin.
- Binding to fibronectin was reduced when P. multocida cells were pre-treated with anti-PmOmpA antibodies.
- PmOmpA binds fibronectin in both native and heat-modified states.
Conclusions:
- PmOmpA is an immunogenic outer membrane protein involved in P. multocida adherence.
- The protein likely facilitates adherence through bridging interactions with host cell fibronectin and/or heparin.
- PmOmpA represents a potential target for therapeutic interventions against P. multocida infections.
