Date

2012

Author

Tefon, Burcu Emine

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Bordetella pertussis is a gram-negative, human pathogen and etiologic agent of whooping cough (pertussis), a highly contagious, acute respiratory illness. In this study, the analysis of whole immunproteome and subproteomes of this microorganism was performed. The soluble cytoplasmic proteomes of B. pertussis Tohama I strain and a local isolate Saadet were separated by 2DE. By Western blot analysis, we identified 25 immunogenic proteins of three categories. In the first group, there were well-known proteins of the pathogen The second group comprised proteins which were already shown antigenic in certain pathogenic bacteria, but not in B. pertussis before. The third group of proteins were those which have not been shown to be immunogenic in any pathogen till the present study such as putative chromosome partition protein, preprotein translocase SecA subunit, carbamoyl-phosphate synthase large chain, PRP synthase, putative substrate-CoA ligase, lysyl-tRNA synthetase, fumaryl acetoacetase, putative peptidyl-prolyl cis-trans isomerase, aspartate-semialdehyde dehydrogenase, putative DNA-binding protein and a putative outer membrane protein. In our surfaceome study, surface proteins of two strains were identified by 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these techniques 45 proteins were identified by 2DE and 226 proteins by geLC-MS/MS. The immunogenicity of surface proteins on 2DE gels were analyzed by Western blotting and among 11 identified immunogenic proteins glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase were found to be immunogenic for the first time in Bordetella. It was also found that 16 proteins were differentially expressed in B. pertussis Saadet and Tohama I. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I. In the secretome study, we identified 40 proteins by 2DE and 357 proteins by geLC-MS/MS. It was found that 12 proteins were immunogenic by Western blot analysis and the immunogenicity of putative secreted protein (BP1047) was shown for the first time in this study. In our study, PT subunit 2 and putative outer protein D (BopD) were more abundant in Saadet while one protein, glutamate synthase subunit beta was expressed at a higher level in Tohama I. Four proteins were expressed only in Saadet (two capsular polysaccharide biosynthesis protein, protein FimX and putative outer membrane permeability protein). The present study comprehensively covered almost the entire proteome of a crucial pathogen, demonstrated many novel antigens and identified hundreds of membrane-bound proteins, cell surface-associated and extracellular proteins. Thus, it is anticipated to greatly aid in a better understanding of pathogen-host relations, rational design of novel drugs and developing new generation vaccines against B. pertussis.

Subject Keywords

Bordetella pertussis., Whooping cough

URI

http://etd.lib.metu.edu.tr/upload/12614238/index.pdf
https://hdl.handle.net/11511/21451

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Graduate School of Natural and Applied Sciences, Thesis