Naturally Secreted Outer Membrane Vesicles of Campylobacter jejuni: Role in Host-Microbe Interaction and Vaccine Perspectives

Khan, Afruja (2025) Naturally Secreted Outer Membrane Vesicles of Campylobacter jejuni: Role in Host-Microbe Interaction and Vaccine Perspectives. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Afruja Khan (19RS107)) 19RS107.pdf - Submitted Version Restricted to Repository staff only Download (31MB)

Abstract

Campylobacteriosis is an acute diarrheal illness and gastroenteritis caused by mainly Campylobacter jejuni (C. jejuni), an opportunistic and hard-to-treat gut pathogen. It remains a significant public health risk in developing countries, with substantial mortality and morbidity in children under the age of five. C. jejuni has a broad host range, but its primary reservoir is poultry, particularly chickens, and transmitted to humans via the consumption of contaminated food and water. In contrast to acute gastroenteritis in humans, C. jejuni exhibits prolonged cecal colonization at a high level with low pathological outcomes in chickens. Genetic diversities among C. jejuni and a limited understanding of immunological correlations of host protection remain challenging for developing an effective measure to control Campylobacter infection. Given the role of bacterial outer membrane-associated proteins in intestinal adherence and invasion, bacterial outer membrane vesicles (OMVs) have emerged as a potential vaccine target against several gut pathogens. Naturally secreted OMVs from gut microbes carry diverse cargo, including proteins, nucleic acids, toxins, and many unidentified secretory factors. Bacterial OMVs can shuttle molecules across different cell types as a generalized secretion system, facilitating bacterial pathogenicity and self-survival. Many mucosal pathogens, including C. jejuni, share various mechanisms of harmonized secretion of major virulence factors. Intriguingly, as a common gut pathogen, C. jejuni lacks some classical virulence-associated secretion systems; alternatively, it often employs nanosized lipid-bound OMVs as an intensive strategy to deliver toxins, including secretory proteins, into the target cells. To better understand how the biophysical and compositional attributes of natural OMVs of C. jejuni regulate their cellular interactions to induce biologically relevant host responses, we performed an in-depth morphological and compositional analysis of naturally secreted OMVs of C. jejuni. Next, we focused on understanding the mechanism of host cell-specific OMV uptake from the extracellular milieu using two different host targets (human and avian cells). This study showed that intracellular perfusion of OMVs is mediated by cytosolic as well as multiple endocytic uptake processes due to the heterogenic nature, abundance of surface proteins, and membrane phospholipids derived from the source bacteria. Further in vitro studies revealed that OMVs can significantly increase C. jejuni invasion and upregulate several proinflammatory cytokines genes. Finally, to analyse the in vivo immune correlates of OMVs, we investigated their mucosal vaccine potential in murine and avian models. To overcome the intrinsic challenges of mucosal delivery of OMVs associated with variable pH and risk of enzymatic degradation, we used chitosan as a nontoxic, mucoadhesive bio-polymer as a protective shield for OMVs. The data presented herein suggest that intragastric delivery of chitosan-coated OMVs (CS-OMVs) confers significant immune protection against C. jejuni with marked local and systemic antibody production in immunized mice. Further, immunization with OMVs resulted in potent cellular responses with increased CD4⁺ and CD8⁺ T cell phenotypes along with marked upregulation of IFN γ and IL-6 gene expression, suggesting that mucosal delivery of OMVs can promote a Th1/Th2 mixed-type immune response in mice. Employing a similar strategy, we next tested the in vivo effects of CS-OMVs immunization in chickens. We demonstrated that oral administration of CS-OMVs can also drive a marked increase in local antibody (sIgA) responses with significant upregulation of pro-inflammatory cytokines (IL-1β, IL-8 and IL-17), leading to effective immunoprotection against a cecal load of C. jejuni. Together, the present study highlights that as an acellular and nonreplicating canonical end product of bacterial secretion, mucosal delivery of OMVs may represent a promising vaccine candidate against C. jejuni.

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