Immunoproteomic analyses of outer membrane proteins ofMannheimia haemolyticaand identification of potential vaccine candidates

Abstract

Immunoproteomic analyses were used to characterize the outer membrane proteome of Mannheimia haemolytica, formerly Pasteurella haemolytica, serotype 1, and determine potential vaccine candidate proteins. 2-DE of M. haemolytica outer membranes was followed by immunoblot analyses using naïve and convalescent bovine sera. Proteins were identified using MALDI-TOF and LC-MS/MS. Spectral data was used to mine M. haemolytica protein database and 132 immunoreactive proteins were identified. Bioinformatic analysis using PSORTb, SubLoc, LipoP, BOMP, MCMBB, and TMB-Hunt/BBTM to predict subcellular localization of immunoreactive proteins and beta-barrels narrowed the list down to 55 candidates. Functional characterization of 55 proteins predicted 16 (29%) are involved in cell structure, 13 (23.6%) in transport/virulence, ten (18.2%) as unknown, six (10.9%) in general metabolism, four (7.27%) in cell process, two (3.64%) in translation, and one (1.8%) each in DNA replication, regulation, transcription, and virulence. Prediction of beta-barrel formation was between 11 and 31 immunoreactive proteins depending on the bioinformatic tool employed. Some of these proteins have potentials to be developed into stand-alone vaccines or components of vaccines. Of those proteins, several have already been characterized. Finally, although characteristics of many of M. haemolytica immunoreactive proteins identified in this study were obtained from published data and predictions using bioinformatics tools, five proteins previously listed in the published M. haemolytica sequence as unidentified were found to have correlates with functional proteins in other bacterial species.

Proteomic Analysis and Immunogenicity of Mannheimia haemolytica Vesicles

“…Mannheimia haemolytica serotype S1, strain 89010807N, originally isolated from a case of calf pneumonia, was used for this study (20). Growth conditions of the bacterium have been described previously (4).…”

“…An immunoproteomic study of M. haemolytica OMPs conducted in our laboratory identified 57 OMPs that may have the potential to be developed into vaccines (4). The immunogenicity of recombinant forms of several M. haemolytica OMPs, including PlpE, OmpA, PlpF, OmpP2, serotype 1-specific antigen (SSA-1), and OmpD15, has been studied (5)(6)(7)(8)(9)(10).…”

Outer Membrane Protein A of Bovine and Ovine Isolates of Mannheimia haemolytica Is Surface Exposed and Contains Host Species-Specific Epitopes

“…Several studies have demonstrated the importance of M. haemolytica OMPs as surface antigens and their potential as vaccine components (3,14,56,57,61). Antibodies against the immunogenic and surface-exposed lipoprotein PlpE contribute toward host defense (60), and the addition of recombinant PlpE to commercial vaccines significantly enhances protection against experimental challenge (12,13).…”

Immunogenicity of Mannheimia haemolytica Recombinant Outer Membrane Proteins Serotype 1-Specific Antigen, OmpA, OmpP2, and OmpD15

“…SSA-1 colony and 2D gel immunoblots with bovine convalescent-phase anti-M. haemolytica sera previously demonstrated high antibody responses to the native protein (4,30). Southern blotting demonstrated genomic fragments homologous to ssa1 in numerous M. haemolytica serotypes, and sequence homology between the ssa1 genes from serotypes 1 and 2 is Ͼ99% (19,21).…”

“…We therefore undertook the process of identifying other OMPs that may be important immunogens. Through immunoproteomic analyses using two-dimensional (2D) gel electrophoresis and Western blots of M. haemolytica outer membrane preparations probed with convalescent-phase cattle sera, we identified several additional OMPs of interest for further study (4). We cloned and expressed the genes for five of these proteins, namely PlpF, serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15, and then purified the proteins.…”