Understanding the Molecular Mechanism of Campylobacter jejuni Pathogenesis for Vaccine Development

Singh, Ankita (2020) Understanding the Molecular Mechanism of Campylobacter jejuni Pathogenesis for Vaccine Development. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Among the major enteric pathogens, Campylobacter jejuni (C. jejuni) is considered as an important source for diarrhoeal illness in humans. In contrast to acute gastroenteritis in humans, C. jejuni exhibits prolonged cecal colonization at a high level with little or no pathology in chickens. Although several putative virulence factors of C. jejuni were found to be associated with a higher degree of pathogenesis in humans, to date, little is known about the basis for their persistent colonization in chickens. To this end, the role of C. jejuni in differential host immune responses using human and chickens cells was assessed. Based on the abundance of major genes encoding virulence factors (GEVFs), the particular isolate that harbors cadF, flaA, peb1, racR, ciaB, cdtB, and hcp genes was used. The study showed that higher bacterial invasion to human intestinal epithelial INT407 cells triggers high-level expression of major pro-inflammatory cytokines such as Interleukin (IL)-1β, IL-6 with significant downregulation of IL-17A gene expression. These observations suggest that the enhanced ability of C. jejuni to invade human cells is tightly regulated by the pro-inflammatory cytokines in the intestine and possibly holds the keys for the observed difference in pathogenesis seen between human and chickens cells. Additionally, the hypervirulent C. jejuni used in this study, which encodes haemolysin co-regulated protein (hcp) gene as a hallmark of functional Type VI Secretion System (T6SS) showed significantly higher adhesion and invasion potential in human INT407 cells compared to primary chickens embryo intestinal cells (CEICs). This observation was supported by Scanning Electron Microscopic (SEM) analysis, which revealed characteristic "attaching and effacing" lesions in human cells caused by 18aM C. jejuni while little or no change was observed in primary CEICs. Thus, the greater ability of hypervirulent C. jejuni isolate to invade INT407 cells and create phenotypic changes in the membrane architecture of human cells suggest the role of hcp in C. jejuni virulence and pathogenicity. Considering the association of hcp with bacterial virulence and host pathogenesis, the possible contribution of hcp in the observed differences in pathogenesis between human and chickens cells was further investigated. The marked transcriptional upregulation of hcp gene expression in C. jejuni recovered from human INT407 cells compared to C. jejuni from primary CEICs suggests the role of C. jejuni hcp in increasing pathogenicity in human cells over primary CEICs. In fact, the data of higher sequence conservation, a predominance of several B-cell epitopes, and immune-reactive ability suggest that the Hcp protein reserves its potential to be considered as an effective vaccine target against C. jejuni. Together with its role in promoting virulence in terms of enhanced cell adherence and invasion, it was hypothesized that effective measures to block hcp protein may reduce C. jejuni colonization in the gut. To this end, in further studies, the immunogenic potential of Hcp of T6SS was examined in chickens with a notion to control pathogen transmission to humans via the food chain. Commercial broiler chickens were explicitly used to test the immune-protective efficacy of recombinant Hcp (rHcp) protein largely because of the fact that chickens are the primary source for C. jejuni infection in humans and persistent cecal colonization significantly contribute to pathogen transmission. Considering the mucosal route is the primary portal for C. jejuni entry, and gut mucosa offers the apposite site for C. jejuni adherence, mucosal (intra-gastric) route as a mode for rhcp delivery using chitosan (CS)-based nanoparticles (NPs) was used. To achieve this goal, the full length coding sequence of the hcp gene from C. jejuni was cloned and expressed in E. coli. Purified rHcp was entrapped in chitosan-sodium tripolyphosphate nanoparticles (CS-TPP NPs) and orally gavaged in chickens. Results suggest that intra-gastric immunization of CS-TPP- rHcp induces a consistent and steady increase in intestinal (sIgA) and systemic antibody (IgY) response against rhcp with a significant reduction in cecal load of C. jejuni. The protection afforded by rHcp associated cellular responses with T helper (Th)1 and Th17 profile in terms of increased expression of Nuclear factor-kappa B (NF-kB), Interferon-gamma (IFN-γ), IL-1β, IL-8, IL-6, and IL-17A genes. Though systemic immunization of rHcp with incomplete Freund's adjuvant (IFA) resulting in a robust systemic (IgY) and local (sIgA) antibody response, mucosal administration of rHcp loaded CS-TPP NPs was found to be superior in terms of bacterial clearance. Altogether, this study provides critical insight into our current understanding of differential host responses against C. jejuni infection among humans and chickens. Moreover, along with revealing some key roles of hcp gene of functional T6SS in C. jejuni virulence, the present study also demonstrate that intra-gastric delivery of rHcp adjuvanted with chitosan has several advantages over the injectable composition and could be a promising vaccine approach to effectively control C. jejuni colonization in chickens.

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