Cysteine dependent conformation heterogeneity of Shigella flexneri autotransporter IcsA and implications in its function

Abstract

Shigella IcsA is a versatile surface virulence factor required for both early and late pathogenesis stages, extracellularly to intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing of the host stimuli by Shigella Type III secretion system (T3SS). Here, we report that the single cysteine residue (C130) near the N-terminus of IcsA passenger has a role in IcsA adhesin activity. We also show that the IcsA passenger (IcsAp) exists in multiple conformations, and the conformation populations are influenced by a central pair of cysteine residues (C375 and C379), which is not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues alters the exposure of IcsA epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA antibody reactivity was restored when the IcsA paired cysteine substitution mutants were expressed in a ∆ipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovers a novel molecular switch empowered by a centrally localised, short-spaced cysteine pair in the Type V autotransporter IcsA that ensures conformational heterogeneity to aid IcsA evasion of host immunity. Importance: Shigella species are the leading cause of diarrheal related death globally by causing bacillary dysentery. The surface virulence factor IcsA which is essential for Shigella pathogenesis is a unique multi-functional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we show that the three cysteine residues in IcsA contribute to the protein’s distinct functions. The N terminus cysteine residue within the IcsA passenger domain plays a role in adhesin function, while a centrally localised cysteine pair provides conformational heterogeneity resulting in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserves biological function in distinct IcsA conformations for cell adhesion, actin-based motility and autophagy escape, providing a potential strategy by which Shigella evade host immunity targeting of this essential virulence factor.