In Silico Identification of Novel Immunogenic Secreted Proteins of Mycoplasma bovis from Secretome Data and Experimental Verification

Abstract

Mycoplasma bovis is a major pathogen, responsible for bovine respiratory diseases worldwide. The present lack of effective control measures leaves cattle owners at considerable perpetual risk of M. bovis outbreaks. In this study, we identified M. bovis secreted immunogenic proteins in silico as potential candidates for novel diagnostic agents and vaccines. We used immunoinformatics to analyze 438 M. bovis proteins previously identified with a label-free proteomics analysis of virulent M. bovis HB0801 (P1) and its attenuated P150 strains. The subcellular localization of these proteins was preliminarily screened and 59 proteins were found to be secreted extracellular proteins. Twenty-seven of these proteins contained a large number of predictive T-cell epitopes presented by major histocompatibility complex (MHC) class I and II molecules. Twenty-two of these 27 proteins had a high number of conformational B-cell epitopes, predicted from the corresponding 3D structural templates, including one unique to P1, two unique to P150, and 19 common to both strains. Five proteins were selected for further validation, and two of these, MbovP274 and MbovP570, were successfully expressed and purified. Both were confirmed to be secretory and highly immunogenic proteins that induced a mouse antibody response, reacted with cattle serum positive for M. bovis infection, and significantly increased the production of interleukin 8 (IL-8), IL-12 and interferon γ (IFN-γ) during the secretion of these three cytokines by both M. bovis mutants of these genes. These results should be useful in the development of novel immunological agents against M. bovis infection.

Comparative Proteomic Analysis of Secretory Proteins of Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides Investigates Virulence and Discovers Important Diagnostic Biomarkers

“…The EF-Tu encoded by the TS60_0188 gene was previously identified as an adhesin in M. hyopneumoniae [ 37 ]. In addition, MbovP274 was confirmed to be a secreted protein and could significantly increase the production of IL-8, IL-12, and IFN-γ [ 38 ]. Considering the limited knowledge on secreted proteins of Mmm and M. bovis , our findings provided significant reference for further identifying potential virulence-associated factors to elucidate mycoplasmal pathogenicity.…”

Regularity of Toll-Like Receptors in Bovine Mammary Epithelial Cells Induced by Mycoplasma bovis

“…IL-8 is a chemokine that recruits neutrophils to swarm to sites of infection with multiple pathogens ( 26 ). IL-8 secretion increased after M. bovis infection ( 27 ). All three M. bovis strains promoted IL-8 overexpression in the present work, an observation consistent with the findings of Zbinden et al ( 28 ) in their own study of the BMEC immune response to M. bovis infection.…”

Proteomics approaches: A review regarding an importance of proteome analyses in understanding the pathogens and diseases

“…Using several bioinformatics tools available as web servers, secretory proteins of M. bovis were recently examined. Two proteins (MbovP274 and MbovP570) were chosen from the secretome data and experimentally confirmed to be immunogenic proteins ( 101 ). Many bacterial pathogens ( C. botulinum, C. defficile, Y. pseudotuberclosis, S. saprophyticus , and Legionella sp.)…”

“…In terms of proteomics, Mycoplasma bovis has been a widely investigated bacterium in recent years. Using entire cell proteins, membrane proteins, and secreted proteins, effective research has recently been published in order to uncover diagnostic markers and vaccine candidates ( 8 , 68 , 101 , 173 ). Profiling core secretome proteins among different strains of pathogenic bacteria might be significant to future studies as supported by the recent core genome studies ( 183 ).…”