Polarity and secretion of Shigella flexneri IcsA: a classical autotransporter

Abstract

The classical autotransporter IcsA is an essential virulence factor for the enteropathogen Shigella flexneri as it provides adherence properties and allows intra- and intercellular spreading in the colonic mucosa. IcsA is an outer membrane surface protein that specifically hijacks host-cell actin recruiting and polymerising complexes allowing actin polymerisation as a form of actin-based motility (ABM). Importantly, since IcsA is localised specifically at one end of the bacterium (the pole), the resulting ABM is unidirectional which is a requirement for efficient S. flexneri dissemination. However, the molecular mechanisms that generate IcsA polarity remain poorly understood. Furthermore, IcsA is a member of the autotransporter family of secreted virulence factors (Type Va). Although many steps in the autotransporter pathway have been elucidated, it is still poorly understood how these diverse proteins are efficiently translocated to the bacterial cell surface. As such, this thesis investigates the two arms of IcsA biogenesis: (1) polar targeting and (2) autotransport. Regarding IcsA polarity, it was identified here that the IcsA-specific outer membrane protease IcsP localises to the septa of dividing S. flexneri and to the opposing pole relative IcsA. The concentration of IcsP was higher at the septum than the pole showing a life cycle dependent distribution of IcsP. This provides the basis of a model where IcsP is important during division of S. flexneri for setting up (and the continued maintenance of) IcsA polarity by the proteolysis of misdirected IcsA. Further, multiple previous reports have suggested that the S. flexneri lipopolysaccharide O-antigen surface structure can influence IcsA polarity by augmenting membrane fluidity or by asymmetric masking of IcsA surface exposure. These notions were tested here resulting in data that clearly refutes these models and agues simply that IcsA exposure is masked symmetrically over the bacterial cell surface. Finally, IcsA itself contains polar targeting (PT) regions that direct it to the pole by an, as yet, unclear mechanism. Examination of these regions revealed that the central PT (cPT) is most important in polarity augmentation and contains critical polarity targeting function residues. Regarding IcsA autotransport, a highly conserved but uncharacterised autotransporter motif was scrutinized for potential biogenesis functions and was designated in this work as the passenger-associated transport repeat (PATR). It was found that the PATR plays a critical role in the efficient secretion of IcsA to the cell surface. Strikingly, bioinformatics analyses revealed that the PATR delineates an important separate autotransporter sub-type with unique functions, composition, and architecture.