Investigation of the IcsA-mediated Shigella flexneri hyper-adherence

Abstract

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface localised protein IcsA, in addition to its role in actin-based motility (ABM) intracellularly, acts as a host cell adhesin through unknown mechanism(s). In this thesis, the role of IcsA in cell adherence was confirmed by using purified IcsA53-740 protein to show blocking of Shigella adherence and direct binding to host cells. A specific region (residues 138 to 148) in IcsA’s functional domain was found to be required for the IcsA mediated adherence, but not for polar localisation and actin-based motility. The purified mutant protein IcsA53-740(Δ138-148) was found to no longer block S. flexneri adherence and had reduced ability to interact with host molecules. Additionally, S. flexneri expressing IcsAΔ138-148 was found with significant defect in both cell adhesion and invasion. This may provide useful information for designing therapeutics for Shigella infection. The conformational change of IcsA’s functional domain which is associated with its adhesin activity was also explored. Through limited proteolysis assay, it was found that the IcsAΔ138-148 was unable to adapt the conformation associated with the hyper-adherent phenotype. The purified IcsA passenger domain was found to have an intramolecular interaction which might be a consequence of IcsA’s conformational change. Identification of HeLa cell receptors for IcsA was also attempted. Mass spec data found that non-muscle myosin heavy chain IIA and IIB might be the potential host binding partners for IcsA. Using a myosin IIA deficient cell line COS-7, S. flexneri was still found to be hyper-adherent, and the use of the anti-myosin IIA antibody also failed to reduce Shigella adherence. Far Western blotting indicated that myosin IIA is the only one of several IcsA interacting molecule. The DOC induced hyper-adherence of S. flexneri was also characterised. It was found that DOC triggers the release of IpaD into culture supernatant and promotes the IcsA self-association activity that might be responsible for the IcsA-dependent biofilm formation. Overall, this work provides further clues and expands our understanding of the adhesin function of IcsA, the potential molecular mechanism of IcsA’s conformational change, and the potential receptor for the Shigella adhesion mediated by IcsA.