Roles of secreted bacterial factors in modulation of host cell signalling

Abstract: Pathogenic bacteria employ several secretion systems to release or inject virulence factors that may alter host cell processes, generate a replicative niche, and aid bacterial survival in adverse environments. This thesis presents my investigations on how bacterial factors can modulate host cell signalling mechanisms. We investigated possible signalling pathways involved in targets of the Vibrio cholerae protein MakA that was found to mediate inhibition of tumour cell proliferation. Caenorhabditis elegans grazing on MakA-producing bacteria revealed that MakA may affect lipid-mediated signalling in the nematodes by affecting the level of PPK-1, a homologue of eukaryotic PIP5K1α. We studied the possible effects of MakA on eukaryotic PIP5K1α in human colon cancer cell lines and found decreased levels of PIP5K1α and pAkt in the lipid-signalling pathway. Immunoblot analyses demonstrated that MakA inhibited cyclin-dependent kinase 1 and increased p27 expression in the colon cancer cells, resulting in G2/M cell cycle arrest. MakA also caused downregulation of Ki67 and cyclin D1, limiting cancer cell proliferation. MakA is the first reported bacterial protein targeting the PIP5K1α lipid signalling pathway, thereby displaying anti-cancer capabilities. We discovered that phosphatidic acid (PA)-mediated MakA binding to host cell plasma membranes generated endomembrane-rich aggregates that caused host target cell autophagy and cytotoxicity. PA binding and cell toxicity by MakA required its N-terminal domain. The MakA genetic determinant is located within a novel pathogenicity island that also encodes the MakB, MakC, MakD, and MakE proteins. In most V. cholerae and Vibrio anguillarum genomes, mak genes form an operon, makCDBAE. The immunoblot analyses showed that wild-type V. cholerae A1552 released the MakA, MakB, and MakE proteins via the flagellum, while a flagellum-deficient mutant released very little or none. Structurally, MakA, MakB, and MakE belong to a superfamily of bacterial alpha-pore-forming toxins. Identification and structural analysis of V. cholerae Mak proteins revealed that the MakA/B/E toxin is common to several pathogenic Vibrionaceae strains, and this previously unrecognised tripartite toxin may increase their fitness and pathogenicity in various environments and host organisms. Bacteria release spherical lipid nanostructures, extracellular membrane vesicles, that may play many biological roles. Previously, Escherichia coli was shown to release physiologically active cytolysin A (ClyA) via outer membrane vesicles (OMVs). ClyA, the first recognised member of the bacterial alpha-pore-forming proteins, has become a model for how oligomerization and pore formation occur in membranes. The clyA gene is cryptic in commensal non-pathogenic E. coli bacteria displaying no cytolytic activity. We found that the sublytic concentration of ClyA released via OMVs by non-pathogenic E. coli profoundly affected host cells. The ClyA+ OMVs were rapidly internalised into colon cancer cells by macropinocytosis and clathrin-mediated, dynamin-dependent endocytosis. The OMV-associated ClyA caused reduced levels of cancer-activating proteins like EZH2, H3K27me3, CXCR4, STAT3, and MDM2 via the EZH2/H3K27me3/miR622/CXCR4 signalling axis. Evidently, sublytic levels of ClyA in OMVs from non-pathogenic E. coli can modulate epigenetics by targeting EZH2 protein stability and we hypothesised that E. coli in colorectal cancer microbiomes may preferentially lack this protein. Given our current understanding of ClyA interactions in cancer cell signalling, it will be intriguing to determine if and how the status of the clyA locus is involved in the aetiology of colorectal cancer.