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Immunoproteomics of Bordetella Pertussis and in Silico Search for Novel Vaccine Candidates and Drug Targets Against Pertussis

Author: ZhuYongZuo
Tutor: ZuoJiaDai;GuoXiaoKui;ZhangShuMin
School: Peking Union Medical College , China
Course: Molecular Genetics
Keywords: Bordetella pertussis Bordetella parapertussis immuoproteomics immunogenic proteins genomics vaccine candidates drug targets DNA microarray
CLC: R378
Type: PhD thesis
Year: 2007
Downloads: 450
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Abstract

Whooping cough or pertussis has led to 350,000 children deaths mainly in developing countries and has afflicted up to 40 million children worldwide per year. The introduction of pertussis vaccines in the 1950s has significantly reduced the morbidity and mortality of the disease. In view of their serious side effects in infants and children, the second-generation acellular vaccines have gradually replaced whole-cell pertussis vaccines during recent years. However, despite high vaccination coverage, whooping cough caused by both B. pertussis (B.p) and B. parapertussis (B.pp) is still a serious public health threat and the incidence of pertussis has continued to rise. the main reasons include: 1. the currently used pertussis vaccines does not produce life-long protection, after 3-5 years, the vaccine-induced immunity would be too weak to protect children; 2. Pertussis vaccines have low efficiency against increasing numbers of B.pp infections. So the protective effectiveness of the currently used pertussis vaccines needs to be significantly improved. Because the pathogenicity and immunological mechanism have been not completely clarified, the current researches focusing on developing novel pertussis vaccines, such as fusion expressin of several protective antigens and pertussis DNA vaccine, which could not be satisfactory. In addition to vaccine prevention, drug therapy is also necessary for controlling whooping cough. Macrolide antibiotics have been the antimicrobial of main choice for treatment or postexposure prophylaxis of pertussis, however, macrolide antibiotics frequently produce a lot of serious reverse reactions especially against infants, so no recent macrolide antibiotics have been licensed for use in infants aged＜6 months by the FDA of the USA. Therefore, there is an urgent need to develop novel vaccine candidates and drug targets against both B.p and B.pp. The immunoproteomics techniques, combining specificity of antibody with high resolving power of 2D and precision of mass spectral analysis, has globally applied to search for vaccine candidates、novel drug targets、diagnostic antigens and clinical biomarker. In this article immunoproteomics techniques were firstly used to screen immunogenic proteins from membrane proteins and total extracellular proteins of Bordetella pertussis. The membrane proteins were enriched using Triton X-114 extraction protocol. The total extracellular proteins were isolated using classic TCA method. By using the sera of immunized infants with whole-cell pertussis vaccine, Western blotting was performed with these proteins. Total 32 unique immunogenic proteins including 11 extracellular proteins and 27 membrane proteins were identified successfully by MADLI-TOF/MS. In total immunogenic proteins, 69KDa protein or peractin、serum resistantance protein and outer membrane porin protein were known pertussis virulence factors; putative outer membrane ligand binding protein was important Bvg-intermediate phase protein; elongation factor Tu/Ts、glyceraldehyde-3-phosphate dehydrogenase and chaperonin GroEL which were identified as globally immunogenic proteins in several pathogens such as H.p、Group A streptococcus and Candida albicans, were also identified in our study. Putative L-lactate dehydrogenas、glutamate dehydrogenase、succinate dehydrogenase、isocitrate dehydrogenas、putative penicillin-binding protein precursor firstly identified as immunogenic proteins of B.pertussis have been validated to have immunogenity in other pathogen immunoproteomic researches. Other novel immunogenic proteins included sulfate-binding protein precursor、putative 2-hydroxyacid dehydrogenase、amino acid-binding periplasmic protein、hypothetical protein BP3575 and BP2818. BP3575 and BP2818 with unknown function and strong immunoreactivity should be paid more attention. Further reseach focusing on these immunogenic proteins will devote to known pathogenicity and immunological mechanism of B.pertussis.In addition to immunoproteomic analysis, this preliminary work also integrates results from in silico analysis, comparative genomic hybridization, global transcriptional profiling and protein-protein interaction (PPI) networks to screen potential vaccine candidates and drug targets from B.p against both B.p and B.pp. Of 3436 open reading frames (ORFs) in B.p, 671 had high transcriptional levels in the B.p GMT-1 and B.pp 12822 strain, and 81 Bvg-activated (up-regulated) B.p genes were also found in the genome of B.pp. Seven hundred and thirty-three unique genes, 671 highly expressed and 81 Bvg-activated, were regarded as the preliminary vaccine candidates. When cytoplasmic proteins, inner membrane proteins (IMPs) with transmembrane topologies＞4, highly variable B.p genes among the 165 B.p and B.pp strains, and human homologs in B.p were subtracted from these 733 candidates, 191 genes remained. These 191 were thus identified as the final vaccine candidates against both B.p and B.pp. They had high transcriptional levels in both species, or were associated with virulence and pathogenesis. Moreover, the proteins encoded by these genes were not only potentially surface-exposed in the bacteria, but also well-conserved among the 165 B.p and B.pp strains. We also constructed two PPI networks of B.p and B.pp. Among the 191 vaccine candidates, 22 proteins that were highly essential in both these networks were regarded as suitable drug targets against the two species.Using the theory of reverse vaccinology, 191 potential cross-protective vaccine candidates against both B.p and B.pp were identified among the 3436 ORFs in the B.p genome. Of these 191 vaccine candidates, 22 with high essentiality can be used as suitable drug targets for both species. Further research focusing on the 191 vaccine candidates could accelerate the development of more effective pertussis vaccines and drug therapy against B.p and B.pp infection in the future.

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