A protein secreted by the Salmonella type III secretion system controls needle filament assembly
- Yale University School of Medicine, United States
- University of Kansas, United States
Figures
Figure 1 with 1 supplement
The T3SS-associated protein OrgC is secreted as an early substrate of the S. Typhimurium type III secretion system encoded within its pathogenicity island 1.
(A) Gene organization of the orgC locus within the S. Typhimurium pathogenicity island 1. (B) Amino acid sequence alignment of OrgC homologs. The sequences used in the alignment are: OrgC (S. …
Figure 1—figure supplement 1
Genetic organization of different T3SS loci encoding OrgC homologs.
https://doi.org/10.7554/eLife.35886.003
Figure 2 with 1 supplement
OrgC is necessary for efficient needle assembly but does not affect needle length.
(A) S. Typhimurium ΔorgC secretes elevated amount of early and reduced amount of middle/late substrates. Proteins in bacterial culture supernatants (c. s.) were concentrated by TCA precipitation and …
Figure 2—figure supplement 1
PrgI secretion profile of S.
Typhimurium wild-type, ΔorgC, ΔinvJ, and ΔorgC/ΔinvJ mutant strains. Culture supernatant from S. Typhimurium or its isogenic mutants were examined by western-blot for the presence of PrgI.
Figure 3
T3SS-mediated secretion of OrgC is required for its function.
(A and B) Removal of its first 21 amino acids (A) or N-terminal addition of MBP (B) prevents the secretion of OrgC. Whole cell lysates (w. c. l.) and culture supernatants (c. s.) of wild-type S. …
Figure 4
OrgC can exert its function when exogenously applied to bacterial cells.
(A) His-OrgC was purified by Ni-affinity, ion exchange, and gel-filtration chromatography analyzed by SDS-PAGE and coomassie blue staining. (B – D) Administration of purified OrgC to bacterial cell …
Figure 5
OrgC interacts with the needle filament protein PrgI and accelerates its in vitro polymerization.
(A) MBP-tagged OrgC or MBP alone were expressed in a ∆orgC S. Typhimurium strain and affinity-purified from whole cell lysates with amylose resin. Bound proteins were eluted with maltose and …
Figure 6 with 4 supplements
NMR titrations of OrgC and PrgI interaction.
(A) Selected 1H-15N peaks of 15N labeled OrgC that showed the largest changes upon titration with unlabeled PrgI. (B) Plot of the weighted chemical shift deviation of 15N OrgC titrated with PrgI. (C)…
Figure 6—figure supplement 1
NMR titrations of OrgC and PrgI.
(A) 15N labeled OrgC titrated with unlabeled PrgI. (B) 15N PrgI titrated with unlabeled OrgC. The titrations were monitored by acquiring 2D 1H-15N HSQC spectra.
Figure 6—figure supplement 2
PrgI residues (Ser39 and Ser52) directly involved in OrgC binding are exclusively conserved in PrgI homologs that have an OrgC partner.
Multiple sequence alignment of different needle proteins from several bacteria carrying T3SSs. Ser39 and Ser52 are marked with filled black arrowheads. NCBI accession numbers for the needle …
Figure 6—figure supplement 3
Stability of the OrgC protein deletion mutants.
Whole cell lysates (w. c. l.) or culture supernatants (c. s.) of S. Typhimurium ΔinvJ ΔorgC mutant strains carrying the empty vector pWSK129 (empty) or expressing the indicated carboxy-terminal …
Figure 6—figure supplement 4
Mutations in the PrgI residues important for its interaction with OrgC do not affect type III secretion.
Proteins in the bacterial culture supernatant were concentrated by TCA precipitation and analyzed by SDS-PAGE, followed by immunoblot using specific antibodies to the indicated proteins.
Figure 7 with 4 supplements
NMR structure of OrgC and structural model for its binding to PrgI in the assembled needle filament.
(A and B) The structured domain of OrgC consists of a 4-helix bundle and the PrgI-binding region is located at the C-terminus of the OrgC domain (A). The surface of the PrgI-binding region of OrgC …
Figure 7—figure supplement 1
Comparison between the 2D proton-nitrogen correlation NMR spectra of (A) full length OrgC and (B) N-terminal truncation construct of OrgC.
https://doi.org/10.7554/eLife.35886.015
Figure 7—figure supplement 2
Secondary (A) 13Cα, (B) 1Hα, and (C) 13Cβ chemical shifts of OrgC identified four helices (α1, α2, α3, and α4) in the structure of OrgC.
https://doi.org/10.7554/eLife.35886.016
Figure 7—figure supplement 3
Superposition of the lowest energy 20 NMR structures of OrgC, (A) showing only the structured 4-helix domain of OrgC from residues 44–130, and (B) shown with the flexible PrgI-binding region, residues 134–141 (red lines and spheres).
https://doi.org/10.7554/eLife.35886.017
Figure 7—figure supplement 4
The topology of the OrgC fold shares similarity only partly with the topology of the FliD D1 domain.
The OrgC fold is depicted similar to the topology fold of FliD as reported by Song et al (Song et al., 2017). Shown in colors are the similarity in the folding topology of OrgC and FliD.
Tables
Key resources table
| Reagent type (species) | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB300 | Nature, 291:238 | wild type | Mouse isolate of SL1344 |
| Strain, strain background strain background (Salmonella enterica serovar Typhimurium) | SB2942 | This study | orgC 3xFlag invC::kan | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2943 | This study | orgC 3xFlag invJ::kan | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2946 | This study | orgC 3xFlag spaS-3xFlag | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2947 | This study | orgC 3xFlag spaSN258A-3xFlag | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2326 | This study | ∆invJ flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2939 | This study | ∆invJ∆orgC flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB3079 | This study | mbp-prgH flhD::tet | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB3275 | This study | mbp-prgH flhD::tet ∆orgC | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB762 | Infect. Immun. 68:2335 | flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB1679 | This study | ∆orgC | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2639 | This study | ∆invJ flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB2944 | This study | ∆orgC flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB3272 | This study | ∆invJ invA::kan flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB3273 | This study | ∆invJ invG::kan flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB3274 | This study | ∆invJ ∆prgI flhD::Tn10 | |
| Strain, strain background (Salmonella enterica serovar Typhimurium) | SB3289 | This study | ∆invJ ∆orgC ∆prgI flhD::Tn10 | |
| Antibody | M2 | Sigma | Mouse monoclonal antibody to the FLAG epitope |
Additional files
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Supplementary file 1
Restraints and structural statistics for 20 NMR structures of OrgC.
- https://doi.org/10.7554/eLife.35886.019
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Supplementary file 2
Bacterial strains used in this study.
- https://doi.org/10.7554/eLife.35886.020
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Supplementary file 3
Plasmids used in this study.
- https://doi.org/10.7554/eLife.35886.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.35886.022
