Modulation of cancer cell signaling by haemagglutinin protease HapA from Vibrio cholerae

Vibrio cholerae releases outer membrane vesicles (OMVs) that package virulence factors, yet how vesicular deliveryreshapes host responses, particularly in cancer cells, remains incompletely defined. This thesis characterises the role of OMVs-associated virulence factors in regulating cellular signalling in epithelial cells. Furthermore, the thesis aims toaddress the antitumour activities of the V. cholerae zinc metalloprotease haemagglutinin protease A (HapA), with emphasison how OMV association modulates potency and mechanism. Using intestinal epithelial monolayers, 3D spheroids, and intestinal organoids, we demonstrate that V. cholerae OMVs canassociated with the epithelial cell membrane and are rapidly internalized through various endocytic mechanisms into theintracellular compartments. The OMV-associated HapA causes disruption of tight- and adherens-junction proteins inpolarized Caco-2 cells, and intestinal organoids. Moreover, the OMV-associated HapA compromises barrier integrity moreefficiently than the purified enzyme. OMV-associated HapA is internalised by epithelial cells via endocytosis. The HapA then selectively degraded tight- and adherens-junction proteins, including claudin, ZO-1, and β-catenin, resulting in loss ofepithelial cohesion. Importantly, compare to the soluble protein, OMV-associated HapA was biologically more active indisruption of epithelial cells.To assess effects on carcinoma cells, we analysed V. cholerae secretome mutants and identified HapA as the principal factorreducing epithelial cancer cell viability. Mechanistically, HapA cleaves protease-activated receptors PAR1 and PAR2 atnoncanonical sites distinct from human proteases, eliciting an early, transient MEK–ERK activation that culminates incaspase-7–dependent apoptosis. We further uncover a PAR-independent mechanism in colorectal carcinoma cells whereby HapA directly uses β-catenin as asubstrate, attenuating Wnt/β-catenin signalling, downregulating Axin2 and Cyclin D1, suppressing tumor cell proliferation,and enforcing G0/G1 arrest. Collectively, these studies define dual antitumour mechanisms for HapA: PAR1/2 reprogramming of MEK–ERK dynamicsand direct attenuation of β-catenin signalling. The findings also establish OMVs as a potent vehicle that enhance proteaseefficacy at epithelial and tumour interfaces. This work highlights therapeutically relevant host–pathogen interactions andsupports the exploration of OMV-based delivery or protease-targeted strategies in cancer therapy.