biostudies-literatureQin JDHAC | National Health and Medical Research CouncilRamaciotti FoundationsUniversity of Adelaidee0341022https://www.ebi.ac.uk/biostudies/studies/S-EPMC9769942biostudies-literatureUnknown10(6)Shigella IcsA is a versatile surface virulence factor required for early and late pathogenesis stages extracellularly and 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 the host stimuli by Shigella Type III secretion system (T3SS). Here, we reported that the single cysteine residue (C130) near the N terminus of the IcsA passenger had a role in IcsA adhesin activity. We also showed that the IcsA passenger (IcsAp) existed in multiple conformations, and the conformation populations were influenced by a central pair of cysteine residues (C375 and C379), which was not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues altered 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 an ΔipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovered a novel molecular switch empowered by a centrally localized, short-spaced cysteine pair in the Type V autotransporter IcsA that ensured 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 multifunctional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we showed that the three cysteine residues in IcsA contributed to the protein's distinct functions. The N-terminal cysteine residue within the IcsA passenger domain played a role in adhesin function, while a centrally localized cysteine pair provided conformational heterogeneity that resulted in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserved biological function in distinct IcsA conformations for cell adhesion, actin-based motility, and autophagy escape, providing a potential strategy by which Shigella evades host immunity and targets this essential virulence factor.Microbiology spectrumCysteine-Dependent Conformational Heterogeneity of Shigella flexneri Autotransporter IcsA and Implications of Its Function.PMC97699422017HIG0119GNT1144046Morona RTotsika MHong YQin JfalseCysteine-Dependent Conformational Heterogeneity of Shigella flexneri Autotransporter IcsA and Implications of Its Function.Shigella IcsA is a versatile surface virulence factor required for early and late pathogenesis stages extracellularly and 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 the host stimuli by Shigella Type III secretion system (T3SS). Here, we reported that the single cysteine residue (C130) near the N terminus of the IcsA passenger had a role in IcsA adhesin activity. We also showed that the IcsA passenger (IcsAp) existed in multiple conformations, and the conformation populations were influenced by a central pair of cysteine residues (C375 and C379), which was not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues altered 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 an ΔipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovered a novel molecular switch empowered by a centrally localized, short-spaced cysteine pair in the Type V autotransporter IcsA that ensured 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 multifunctional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we showed that the three cysteine residues in IcsA contributed to the protein's distinct functions. The N-terminal cysteine residue within the IcsA passenger domain played a role in adhesin function, while a centrally localized cysteine pair provided conformational heterogeneity that resulted in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserved biological function in distinct IcsA conformations for cell adhesion, actin-based motility, and autophagy escape, providing a potential strategy by which Shigella evades host immunity and targets this essential virulence factor.2022-01-01T00:00:00Z2022 Dec2026-05-28T21:31:29.665Z2025-02-19T01:07:12.655ZS-EPMC97699423637410610.1128/spectrum.03410-22