Studying Targeted Microbial Dysbiosis of Campylobacter jejuni via Bacterial Type VI Secretion System (T6SS)

Gupta, Subhadeep (2024) Studying Targeted Microbial Dysbiosis of Campylobacter jejuni via Bacterial Type VI Secretion System (T6SS). PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Subhadeep Gupta (18RS026)) 18RS026.pdf - Submitted Version Restricted to Repository staff only Download (32MB)

Abstract

The bacterial type VI secretion system (T6SS) has emerged as a new paradigm of secretion machinery in several Gram-negative pathogens and plays a critical role in translocating effectors molecules into surrounding targets. As a contact-dependent secretion system, T6SS transports a range of effector molecules across the membranes of the target cells. As a common gut pathogen, Campylobacter jejuni (C. jejuni) harbours the T6SS that injects toxic effectors into neighbouring cells, modulating microbial competition in the harsh gut environment. The presence of T6SS has recently been identified in C. jejuni and has been shown to promote the competitive advantage of C. jejuni over competing microorganisms and adaptation to environmental perturbation. Since microbial co-existence in the gut largely relies on interbacterial interactions, in the present study, we investigated the role of T6SS of C. jejuni towards understanding its role in host-microbes or microbe-microbe interactions in a landscape composition of the gut environment. Here, we report results from a measurable experiment in which C. jejuni was used as a predator and E. coli as a prey under relevant biological conditions. We showed evidence for T6SS-mediated killing of E. coli by T6SS-positive C. jejuni, while T6SS-negative C. jejuni failed to target them. Since, T6SS functionality, as well as predator fitness, often gets affected by the landscape context of the host gut and its physiochemical composition, such as pH, bile salt concentration, and the presence of other antimicrobial components, we further asked how the altered gut environment can affect T6SS functionality. To this end, we showed that T6SS activity could entail a cost during bacterial predation under environmental stress using bile salt as a natural stressor. We demonstrated that the enhanced bile salt influx promotes higher reactive oxygen species (ROS) accumulation and subsequent DNA damage due to the prey-driven activation of the T6SS. We also observed that prey-driven T6SS activity could cause "ecological suicide" in C. jejuni under bile salt stress. Further, we confirmed this using an isogenic mutant of C. jejuni that lacks the hemolysin- coregulated protein (hcp), a major effector of T6SS. Intracellular tracking of fluorophore- conjugated bile salts suggested T6SS-mediated bile salt influx into C. jejuni can induce "self- destruction" due to enhanced intracellular oxidative stress. For better understanding, we combined experiments and mathematical modelling to explore how the stress-induced “predation cost” determines ecological outcomes. Consistent with a population-dynamics model, we found predator extinction above a critical bile salt concentration and prey-predator coexistence below this level.Given this, we next explored whether we can utilize the predation cost to develop an effective strategy to control C. jejuni infection of host cells. The targeted depletion of potential gut pathogens is often challenging because of their intrinsic ability to thrive in harsh gut environments. Here, we exploited microbial competition via the bacterial T6SS to selectively reduce the C. jejuni load inhabiting chicken cecum. We further investigated whether the prey- driven activity of T6SS perturbs the in vivo colonization of C. jejuni. Using chickens as primary hosts of C. jejuni, we showed effective intestinal clearance of T6SS-positive C. jejuni in chickens with orally administered bile salt solution. Further, analysis of local antibody responses and pro-inflammatory gene expression showed a reduced risk of tissue damage, indicating that T6SS-dependent predation can be utilized as a possible measure to clear the persistent colonization of C. jejuni in chickens. Together, in the present thesis, we elucidate how C. jejuni uses its T6SS to deploy toxic effectors effectively and eliminate other bacterial strains. In conclusion, based on our observation, we propose that the functional T6SS can trigger the intracellular influx of bile salts, causing ROS accumulation and DNA damage of C. jejuni and ultimately resulting in complete elimination. Thus, the unique feature of T6SS can serve as an invaluable tool for selective targeting of T6SS-bearing gut pathogens and can improve gut health.

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