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Cited by 6Escherichia coli research studiesBiochemical and Structural CharacterizationBacterial Genetics and Biotechnology

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Cysteine-Dependent Conformational Heterogeneity of Shigella flexneri Autotransporter IcsA and Implications of Its Function

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Qin, Jilong, Hong, Yaoqin, Morona, Renato, Totsika, Makrina·2022·Microbiology Spectrum
DOI 10.1128/spectrum.03410-22PMC PMC9769942

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Abstract

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.

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Figures

FIG 1 The autotransporter protein IcsA passenger domain. (A) Schematic representation of defined subregions on IcsA passenger affecting its known biological functions with all three cysteine positions shown. PT, polar targeting. (B) IcsA passenger tertiary structure predicted by AlphaFold (AF-Q7BCK4-F1) with defined functional subregions marked in color as in (A) and cysteine residue locations is shown in a close-up view. GRR, glycine-rich repeats (aa 117 to 307).
FIG 1 The autotransporter protein IcsA passenger domain. (A) Schematic representation of defined subregions on IcsA passenger affecting its known biological functions with all three cysteine positions shown. PT, polar targeting. (B) IcsA passenger tertiary structure predicted by AlphaFold (AF-Q7BCK4-F1) with defined functional subregions marked in color as in (A) and cysteine residue locations is shown in a close-up view. GRR, glycine-rich repeats (aa 117 to 307).CC BY · PubMed Central
FIG 2 Cysteine substitutions in IcsAp did not affect surface display but influenced protein conformation. (A) Western immunoblotting of bacterial cell lysates expressing IcsAWT and cysteine mutant detected with anti-IcsA and anti-SurA antibodies. IcsAFL, IcsA full-length. (B) Indirect bacterial surface immunofluorescent labeling of S. flexneri expressing IcsAWT and cysteine substitution mutants with anti-IcsA antibodies. (C) Western immunoblotting of IcsA with lysates of bacterial cells with and without proteinase K surface shaving. (D) Western immunoblotting of precipitated extracellular cleaved IcsAp with anti-IcsA and anti-IpaD antibodies. (E) Western immunoblotting of lysates of bacterial cells expressed FLAG-tagged IcsAWT and cysteine substitution mutants with anti-FLAG and anti-SurA antibodies. (F) Indirect bacterial surface immunofluorescent labeling of FLAG-tagged IcsA passenger with anti-IcsA antibodies and anti-FLAG antibody. Scale bars are shown as 2 μm in (B and F).
FIG 2 Cysteine substitutions in IcsAp did not affect surface display but influenced protein conformation. (A) Western immunoblotting of bacterial cell lysates expressing IcsAWT and cysteine mutant detected with anti-IcsA and anti-SurA antibodies. IcsAFL, IcsA full-length. (B) Indirect bacterial surface immunofluorescent labeling of S. flexneri expressing IcsAWT and cysteine substitution mutants with anti-IcsA antibodies. (C) Western immunoblotting of IcsA with lysates of bacterial cells with and without proteinase K surface shaving. (D) Western immunoblotting of precipitated extracellular cleaved IcsAp with anti-IcsA and anti-IpaD antibodies. (E) Western immunoblotting of lysates of bacterial cells expressed FLAG-tagged IcsAWT and cysteine substitution mutants with anti-FLAG and anti-SurA antibodies. (F) Indirect bacterial surface immunofluorescent labeling of FLAG-tagged IcsA passenger with anti-IcsA antibodies and anti-FLAG antibody. Scale bars are shown as 2 μm in (B and F).CC BY · PubMed Central
FIG 3 The IcsA N terminus had the altered conformation and was maintained in the cleaved IcsAp. (A) Western immunoblotting with bacterial cells expressing IcsA and its cysteine substitution mutants treated with hNE. Samples were taken at different time points as indicated above the blot. im, immediate recovery of the digestion sample upon the addition of hNE. Samples from bacteria expressing IcsAWT were electrophoresed in parallel (left) with mutants (right) and blotted together to ensure equal exposure for direct comparison. (B) Western immunoblotting of fractionated hNE digestion samples from the bacteria expressing IcsAWT. (C) Model of the digestion differences due to altered conformation in IcsA mutant with estimated cutting sites shown. (D) Western immunoblotting of IcsAp separated in semidenatured conditions. Extracellular IcsA was precipitated in ammonium sulfate and was solubilized in SDS-PAGE buffer in the presence or absence of DTT and was subjected to SDS-PAGE and immunoblotting without heating. The underexposure image in the red border correlates to the region in the long exposure image marked red. re, reduced; ox, oxidized. <, IcsAp dimer; blue arrow, oxidized IcsAC375S, C375S-ox. (E) Culture supernatant IcsAp protein recovered as above was analyzed by Native-PAGE and detected with anti-IcsA antibodies. Green arrow, oxidized IcsAp, IcsAp-ox.
FIG 3 The IcsA N terminus had the altered conformation and was maintained in the cleaved IcsAp. (A) Western immunoblotting with bacterial cells expressing IcsA and its cysteine substitution mutants treated with hNE. Samples were taken at different time points as indicated above the blot. im, immediate recovery of the digestion sample upon the addition of hNE. Samples from bacteria expressing IcsAWT were electrophoresed in parallel (left) with mutants (right) and blotted together to ensure equal exposure for direct comparison. (B) Western immunoblotting of fractionated hNE digestion samples from the bacteria expressing IcsAWT. (C) Model of the digestion differences due to altered conformation in IcsA mutant with estimated cutting sites shown. (D) Western immunoblotting of IcsAp separated in semidenatured conditions. Extracellular IcsA was precipitated in ammonium sulfate and was solubilized in SDS-PAGE buffer in the presence or absence of DTT and was subjected to SDS-PAGE and immunoblotting without heating. The underexposure image in the red border correlates to the region in the long exposure image marked red. re, reduced; ox, oxidized. <, IcsAp dimer; blue arrow, oxidized IcsAC375S, C375S-ox. (E) Culture supernatant IcsAp protein recovered as above was analyzed by Native-PAGE and detected with anti-IcsA antibodies. Green arrow, oxidized IcsAp, IcsAp-ox.CC BY · PubMed Central
FIG 4 Purified IcsA existed in two different conformations. (A) Affinity purified IcsAp resolved by SDS-PAGE and stained with Coomassie blue. (B) SEC separation of IcsAp into populations I and II. (C) SDS-PAGE of IcsAp populations I and II isolated by SEC. (D) Native-PAGE of IcsAp populations I and II isolated by SEC. (E) SDS-PAGE of IcsAp cross-linked with 0.1 mM DSP in the presence or absence of 10 mM DTT. The image was from the same gel with the relevant area shown.
FIG 4 Purified IcsA existed in two different conformations. (A) Affinity purified IcsAp resolved by SDS-PAGE and stained with Coomassie blue. (B) SEC separation of IcsAp into populations I and II. (C) SDS-PAGE of IcsAp populations I and II isolated by SEC. (D) Native-PAGE of IcsAp populations I and II isolated by SEC. (E) SDS-PAGE of IcsAp cross-linked with 0.1 mM DSP in the presence or absence of 10 mM DTT. The image was from the same gel with the relevant area shown.CC BY · PubMed Central
FIG 5 Effect of cysteine substitution mutation on IcsA function. (A) Cell adhesion assay of indicated S. flexneri strains with HeLa cells. Mean values from 6 independent assays with standard error mean (SEM) are shown. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test against ΔipaDΔicsA pIcsAWT. ****, P < 0.0001. (B) HeLa cell invasion assay of indicated S. flexneri strains treated with 2.5 mM DOC. Data represent 3 independent assays. (C) Fluorescent labeling of host cell F-actin by phalloidin and host and bacterial nuclei by DAPI of HeLa cells infected with indicated S. flexneri strains. Scale bar is 10 μm. (D and E) Plaque sizes formed by indicated S. flexneri strains in MDCK-2 monolayers. Data were from two independent assays with ~30 plaque sizes plotted for each strain.
FIG 5 Effect of cysteine substitution mutation on IcsA function. (A) Cell adhesion assay of indicated S. flexneri strains with HeLa cells. Mean values from 6 independent assays with standard error mean (SEM) are shown. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test against ΔipaDΔicsA pIcsAWT. ****, P < 0.0001. (B) HeLa cell invasion assay of indicated S. flexneri strains treated with 2.5 mM DOC. Data represent 3 independent assays. (C) Fluorescent labeling of host cell F-actin by phalloidin and host and bacterial nuclei by DAPI of HeLa cells infected with indicated S. flexneri strains. Scale bar is 10 μm. (D and E) Plaque sizes formed by indicated S. flexneri strains in MDCK-2 monolayers. Data were from two independent assays with ~30 plaque sizes plotted for each strain.CC BY · PubMed Central
FIG 6 Effect of ΔipaD on IcsA conformation. (A) Indirect bacterial surface immunofluorescent labeling of FLAG-tagged IcsA passenger with anti-IcsA antibodies and anti-FLAG antibody. (B) Western immunoblotting of IcsAp recovered from the culture supernatant of indicated S. flexneri strains expressing IcsA and IcsA mutants in nondenatured conditions. Samples were solubilized in SDS-PAGE buffer with or without DTT and subjected to SDS-PAGE and Western immunoblotting without heating. The underexposure image in the red border correlates to the region in the long exposure image marked red. re: reduced, ox: oxidized, <: IcsAp dimer; blue arrow: oxidized IcsAC375S, C375S-ox. (C) IcsAp protein recovered as above and analyzed by Native-PAGE and detected with anti-IcsA antibodies. Green arrow: oxidized IcsAp, IcsAp-ox.
FIG 6 Effect of ΔipaD on IcsA conformation. (A) Indirect bacterial surface immunofluorescent labeling of FLAG-tagged IcsA passenger with anti-IcsA antibodies and anti-FLAG antibody. (B) Western immunoblotting of IcsAp recovered from the culture supernatant of indicated S. flexneri strains expressing IcsA and IcsA mutants in nondenatured conditions. Samples were solubilized in SDS-PAGE buffer with or without DTT and subjected to SDS-PAGE and Western immunoblotting without heating. The underexposure image in the red border correlates to the region in the long exposure image marked red. re: reduced, ox: oxidized, <: IcsAp dimer; blue arrow: oxidized IcsAC375S, C375S-ox. (C) IcsAp protein recovered as above and analyzed by Native-PAGE and detected with anti-IcsA antibodies. Green arrow: oxidized IcsAp, IcsAp-ox.CC BY · PubMed Central

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Metadata

Authors
Qin, J., Hong, Y., Morona, R., & Totsika, M.
Affiliations
  • Queensland University of Technology (AU)
  • The University of Adelaide (AU)
Corresponding author
  • Qin, Jilong
  • Totsika, Makrina
ORCID
Venue
Microbiology Spectrum
Publisher
American Society for Microbiology
Published
2022-12-21
Volume
10
Issue
6
Pages
e0341022-e0341022
ISSN
2165-0497 (ISSN-L)
DOI
10.1128/spectrum.03410-22
PMC
PMC9769942
OpenAlex
W4308957989
Cited by
6
References
49
Citations by year
2025: 1 · 2024: 2 · 2023: 2 · 2022: 1
FWCI
0.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbiology Spectrum (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ResearchOnline - JCU (James Cook University) (submittedVersion)
  • Adelaide Research & Scholarship (AR&S) (University of Adelaide) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • QUT ePrints (Queensland University of Technology) (submittedVersion)
Funders
  • DHAC | National Health and Medical Research Council (GNT1144046)
  • Ramaciotti Foundations (2017HIG0119)
  • University of Adelaide
Topics
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
Shigella flexneri, IcsA, autotransporters, shigellosis, cell adhesion, actin-based motility, protein conformation
MeSH terms
Type III Secretion Systems, Type V Secretion Systems, Actins, Bacterial Proteins, Cysteine, DNA-Binding Proteins, Shigella, Shigella flexneri, Transcription Factors, Adhesins, Bacterial, Virulence Factors

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