Studying the Molecular Basis of Campylobacter jejuni Host Cell Adhesion Towards Designing Multicomponent Mucosal Vaccine

Biswas, Prakash (2026) Studying the Molecular Basis of Campylobacter jejuni Host Cell Adhesion Towards Designing Multicomponent Mucosal Vaccine. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Prakash Biswas (20RS106)) 20RS106.pdf - Submitted Version Restricted to Repository staff only Download (16MB)

Abstract

Diarrheal diseases remain a leading cause of preventable death among children under the age of five globally, accounting for an estimated 2-3 million deaths annually, and are highly prevalent in low- and middle-income countries (LMICs). Among bacterial pathogens causing diarrheal illness, Campylobacter jejuni (C. jejuni) remains a major contributor. In contrast to the acute gastroenteritis in humans, C. jejuni establishes prolonged cecal colonization in chickens with little or no associated pathology, making poultry a primary reservoir for human infection. Among the key pathogen-associated factors, several surface-expressed colonization proteins (SECPs)-including Campylobacter adhesion to fibronectin (CadF), fibronectin-binding protein A (FlpA), and jejuni lipoprotein A (JlpA)-as well as secretion system-associated proteins such as hemolysin co-regulated protein (Hcp) and valine-glycine repeat protein G (VgrG), and exotoxins such as cytolethal distending toxins (CDTs), have been implicated in bacterial adhesion and host cell invasion. However, the mechanisms underlying the distinct outcomes of C. jejuni infection in human versus avian hosts remain unclear, particularly with respect to host-specific interactions mediated by cell adhesion proteins of C. jejuni. In the first part of this study, we investigated the role of the key adhesion protein CadF in mediating host-pathogen interactions using an integrated approach combining in silico, in vitro, and in vivo analyses. Employing the cadF mutant of C. jejuni (ΔcadF C. jejuni), we confirmed a marked decrease in C. jejuni adhesion and invasion of both human and avian cells, which led us to investigate further the differential binding of CadF to a common cellular receptor, fibronectin (FN), a key component of the extracellular matrix (ECM). An analysis of molecular docking suggests that the Group 3C domains of human and chicken FN bind exclusively to the FRLS motif (Phenylalanine-Arginine-Leucine-Serine) of CadF. These binding predictions were further confirmed experimentally using the recombinant CadF protein, indicating relatively higher binding of CadF to the chicken FN 3C domain than to the human FN 3C domain. Accordingly, we interpret this result as indicating that the increased binding of CadF to chicken fibronectin likely enhances the ability of C. jejuni to colonize the chicken gut. As a conserved protein across C. jejuni strains, CadF binds to cellular fibronectin (FN), triggering phosphorylation of focal adhesion kinase (FAK) and Src, and inducing actin reorganization that facilitates bacterial invasion and subsequent host cell pathogenesis. Therefore, in the second part of this study, we aimed to develop a prophylactic strategy to inhibit CadF-mediated adhesion to host cells and to neutralize C. jejuni cecal colonization in chickens. However, given that C. jejuni adhesion to the chicken gut is a multifactorial process, a strategy that includes multiple immunogenic subunits would be critical for achieving optimal vaccine efficacy at the mucosal surface. To this end, we envisaged evaluating the vaccine potential of major C. jejuni virulence determinants, including CadF, JlpA, and Hcp, either alone or in combination. To achieve an effective immune response in the gut mucosa, we engineered a biopolymer-coated food-grade, lactic acid-producing (LAB), non-colonizing probiotic vector, Lactococcus lactis subsp. cremoris (strain MG1363), to deliver the target proteins. However, since most LAB vectors are non-commensal and have short gut transit times, we wanted to optimize the LAB vector's gut retention time before an in vivo vaccine study by coating it with a known biopolymer, chitosan. Chitosan is deacetylated from chitin, the second-most-abundant polysaccharide, known for its wide range of uses as a coating polymer. We show that coating rL. lactis expressing reporter proteins (mCherry and mEGFP) with a chitosan polymer cross-linked with tripolyphosphate (TPP) retains its ability to stably express reporter proteins without altering the specificity and sensitivity of fluorescence detection in vitro and in vivo. Further, we provide evidence of enhanced intragastric stability by chitosan-TPP (CS) coating of rL. lactis cells, allowing us to study the spatiotemporal distribution in the chicken gut for an extended period. Our in vivo chicken immunization and infection model revealed that intragastric administration of CS-coated recombinant L. lactis expressing CadF, JlpA, and Hcp induced potent mucosal (sIgA) and cellular immune responses, marked by increased sIgA secretion and pro-inflammatory cytokine expression, which correlated with a marked reduction in C. jejuni cecal colonization. Moreover, we confirmed that this vaccine modality, based on a bioengineered lactic acid bacteria (LAB) vector, also modulates the overall gut microbiota composition, promoting a healthier microbial balance in the poultry gut. Together, this study underscores the role of CadF in differential host cell adhesion and highlights the potential of a live vector-based, multicomponent mucosal vaccine strategy to reduce C. jejuni cecal colonization, thereby potentially lowering foodborne transmission to humans.

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