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Envelope stress responses defend against type six secretion system attacks independently of immunity proteins

Nature Microbiology volume 5, pages 706–714 (2020)Cite this article

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Abstract

The arms race among microorganisms is a key driver in the evolution of not only the weapons but also defence mechanisms. Many Gram-negative bacteria use the type six secretion system (T6SS) to deliver toxic effectors directly into neighbouring cells. Defence against effectors requires cognate immunity proteins. However, here we show immunity-independent protection mediated by envelope stress responses in Escherichia coli and Vibrio cholerae against a V. cholerae T6SS effector, TseH. We demonstrate that TseH is a PAAR-dependent species-specific effector highly potent against Aeromonas species but not against its V. cholerae immunity mutant or E. coli. A structural analysis reveals TseH is probably a NlpC/P60-family cysteine endopeptidase. We determine that two envelope stress-response pathways, Rcs and BaeSR, protect E. coli from TseH toxicity by mechanisms including capsule synthesis. The two-component system WigKR (VxrAB) is critical for protecting V. cholerae from its own T6SS despite expressing immunity genes. WigR also regulates T6SS expression, suggesting a dual role in attack and defence. This deepens our understanding of how bacteria survive T6SS attacks and suggests that defence against the T6SS represents a major selective pressure driving the evolution of species-specific effectors and protective mechanisms mediated by envelope stress responses and capsule synthesis.

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Fig. 1: TseH is an NlpC/P60 peptidase delivered by PAAR2 that kills A. dhakensis but not E. coli or V. cholerae.
Fig. 2: Envelope stress responses protect E. coli against T6SS-mediated killing.
Fig. 3: V. cholerae WigR is necessary to survive T6SS self-killing independently of immunity genes.
Fig. 4: The protective role of WigKR supersedes VPS expression.
Fig. 5: WigR regulates the T6SS in V52.
Fig. 6: Envelope stress responses defend against T6SS system attack.

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Data availability

Source data for Figs. 1–5 and Extended Data Figs. 1 and 3–5 are provided with the paper. Final structural model was deposited to the PDB (accession no. 6V98). Further information is available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and Canadian Natural Sciences and Engineering Research Council (NSERC) to T.G.D., and in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract nos HHSN272201200026C and HHSN272201700060C to A.S. T.G.D. is also supported by a Government of Canada Research Chair award and a Canadian Foundation for Innovation grant (CFI-JELF). A.P. was funded by the Markin Undergraduate Student Research Program in Health and Wellness. L.L. is supported by an Alberta Innovates Health Solutions (AIHS) graduate student scholarship and K.M. by an Alberta Queen Elizabeth II Graduate Scholarship. B.B. was supported by AIHS and National Sciences and Engineering Research Council of Canada (NSERC) postdoctoral fellowships. S.J.H. holds a CIHR postdoctoral fellowship. We thank Z. Eltsova for assistance in obtaining Keio and ASKA collection strains as well as W. Navarre for helpful discussions.

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Authors and Affiliations

  1. Department of Ecosystem and Public Health, University of Calgary, Calgary, Canada

    Steven J. Hersch, Maria Silvina Stietz, Kevin Manera, Fatima Kamal, Brianne Burkinshaw, Linh Lam, Alexander Pun, Meixin Li & Tao G. Dong

  2. Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Canada

    Nobuhiko Watanabe & Alexei Savchenko

  3. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Tao G. Dong

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Contributions

S.J.H. designed, performed and analysed most of the biological experiments and prepared the manuscript and figures. N.W. and A.S. performed and analysed the crystallography. M.S.S. performed the microscopy. K.M. performed and analysed the RT–qPCR. F.K. performed the permeability analysis, helped construct plasmids and identified reduced-toxicity TseH mutants. B.B. performed and analysed the pull-down assays. L.L. analysed the genome-sequencing data. A.P. and M.L. helped construct the strains and plasmids. N.W., A.S. and T.G.D. contributed to the manuscript revision. T.G.D. conceived the project and supervised the study.

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Correspondence to Tao G. Dong.

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Extended data

Extended Data Fig. 1 Further analyses of TseH killing, delivery, and structure.

a. Relative survival (killing by V. cholerae V52 TseHWT / TseHH64A) of E. coli, A. hydrophila, Edwardsiella sp., A. baylyi ADP1 (ΔT6SS), or V. parahaemolyticus RIMD2210633 strains. Welch’s one-way ANOVA with Dunnett’s multiple comparisons test comparing each sample to E. coli MG1655; *, p < 0.05; **, p < 0.01; ns, not significant. b. Relative survival (killing by TseHWT strain relative to killing by TseHH64A strain) of A. dhakensis expressing TsiH or vector only control. Unpaired 2-tailed t-test; ***, p < 0.001. c. Survival of A. dhakensis after killing by TseHH64A strain expressing TseH or vector only. Vector control data resembles no-plasmid data shown in Fig. 1a and was only repeated in duplicate. Unpaired 2-tailed t-test with Welch’s correction; **, p < 0.01. d. Extension of Fig. 1b in the main text. Survival of A. dhakensis prey after killing by ΔPAAR2 strain expressing plasmid-borne TseH and full-length or C-terminal truncations of PAAR2. For PAAR2: V, vector; numbers, PAAR2 truncated to indicated amino acid length; Δin, codons 146–156 removed leaving downstream residues intact. One-way ANOVA with Dunnett’s multiple comparisons test comparing to the sample expressing TseH and Vector control of PAAR2; ***, p < 0.001; ns, not significant. For all graphs, the mean and standard deviation are shown. Dots show individual replicates. e. Western blot showing whole cell lysate or eluted fraction after His-tag pull-down. BL21 DE3 E. coli expressed Flag-tagged TseH in combination with either His-tagged PAAR2 or Spy as a non-binding control. Number at left indicated ladder band size in kDa. Data is representative of two independent replicates. f. Circular permutation of catalytic residues causes swapping of N- and C-terminal lobes in the structure of TseH compared to Tse1 (PDB: 4EOB18), the other T6SS effector that belongs to NlpC/P60 family has reversed N- and C-lobe structure in comparison to TseH. g. Restricted access to the catalytic cysteine in certain peptidoglycan endopeptidases. The surface representations of SaCwlT and TseH show that the conformational changes in residues surrounding the active site are necessary for the substrate binding21,22. For all graphs, the mean and standard deviation are shown. Dots show individual replicates. TseHWT, V. cholerae V52 with all anti-bacterial effectors inactivated except TseH; TseHH64A, V52 with all anti-bacterial effectors inactivated including TseH.

Source data

Extended Data Fig. 2

Identified mutations that reduce TseH toxicity.

Extended Data Fig. 3 Additional E. coli mutants susceptible to T6SS-delivered TseH.

a. Disk diffusion induction of plasmid-borne TAT-tagged TseH expression in bacterial lawns of E. coli MG1655. WT (top) or H64A mutant (bottom) TseH are compared. Right discs contain the inducer of expression, arabinose (Ara), and left discs contain the repressor, glucose (Glu). Representative of three independent replicates. b. As in a, comparing induction of plasmid-borne TAT-tagged TseHWT expression in E. coli MG1655 or A. dhakensis (ΔT6SS). Similar data for TseHWT in E. coli has been shown previously and is shown again here for comparison4. c. Relative survival (killing by TseHWT / TseHH64A) of E. coli WT or knockout mutants from indicated categories. One-way ANOVA with Dunnett’s multiple comparisons test comparing each sample to WT; **, p < 0.01; ***, p < 0.001; ns, not significant. Data for WT (E. coli) is shown in Fig. 2 and is shown again here for comparison. Aux., auxotrophs. d. RT-qPCR comparing expression of genes in WT E. coli prey after T6SS competition assay with TseHWT or TseHH64A killer strains. Value with TseHH64A killer was set to 1 to show fold induction in response to TseH activity upon delivery by the T6SS. One-way ANOVA (with samples shown in Fig. 2d) with Sidak’s multiple comparisons test; ns, not significant. For both graphs, the mean and standard deviation are shown. Dots show individual replicates. TseHWT, V. cholerae V52 with all anti-bacterial effectors inactivated except TseH; TseHH64A, V52 with all anti-bacterial effectors inactivated including TseH.

Source data

Extended Data Fig. 4 Additional data for RcsA and BaeR regulon overexpression, and effect of Spy overexpression on permeability.

a. Disk diffusion induction of plasmid-borne TAT-tagged TseH in bacterial lawns of E. coli BW25113 containing RcsA or vector only plasmids. IPTG was added throughout the lawn to induce RcsA. Discs contain arabinose to induce TAT-TseH expression. Representative of three independent replicates. b. Relative survival (killing by TseHWT / TseHH64A) of E. coli ΔdegP mutant or A. dhakensis with vector (Vec) or overexpressing genes from the BaeR regulon. One-way ANOVA with Sidak’s multiple comparisons test comparing samples to their respective empty vector controls; *, p < 0.05; **, p < 0.01; ns, not significant. Vector controls are shown in Fig. 2 and are shown again here for comparison. Mean and standard deviations are shown. Dots show individual replicates. pSpyΔSec, plasmid expressing Spy with its Sec secretion tag deleted. c. Images of ΔbaeR E. coli overexpressing plasmid-borne TAT-TseH and either Spy or vector-only controls. The phase-contrast and propidium iodide(PI) channels are merged. Scale bar shows 5 μm. Representative of three independent replicates. d. Quantification showing the percent of total cells showing PI + fluorescence. N number at top indicates total cells counted. Dots show mean results from three independent experiments (used to calculate statistics) and error bars show one standard deviation. Unpaired 2-tailed t-test, p = 0.0513.

Source data

Extended Data Fig. 5 Additional data for ΔwigR strain T6SS susceptibility, immunity gene expression, VPS-mediated aggregation, and Aeromonas species do not encode RcsCDB, BaeSR, or WigKR.

Relative survival (WT V52 / TseHH64A killer strain) of V. cholerae V52 (T6SS+ background) prey (a.) or V. cholerae C6706 prey (b.) with intact (+), deleted (Δ), or complemented (Δ,p) wigR. Stationary (Stat.) and log phase prey are shown. One-way ANOVA with Sidak’s multiple comparisons test; ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant. c. RT-qPCR measuring expression of T6SS immunity genes in ΔwigR V. cholerae V52 relative to in the wigR + strain (both in the ΔT6SS background). For all graphs, the mean and standard deviation are shown. Dots show individual replicates. d. Image showing settling of V. cholerae V52 grown at 37 °C or at 30 °C to induce VPS synthesis. Image after vortexing to disrupt aggregates is also shown for comparison (lower panel). Representative of three independent replicates. e. KEGG ortholog analysis across Gammaproteobacteria. KEGG Modules were compared for RcsCDB and BaeSR. WigK and WigR orthologs were assessed by sequence homology to VCA0565 and VCA0566 using an SW-score cutoff of 50% of maximum. Data is shown as the percentage of strains within each taxonomic group that contain an ortholog of all genes in the loci. Number in brackets indicates the number of strains in the taxonomic group in the KEGG database.

Source data

Extended Data Fig. 6

Table of data collection, phasing and refinement statistics for MAD (SeMet) structures.

Supplementary information

Supplementary Information

Supplementary Table 1: Strains and plasmids.

Reporting Summary

Supplementary Video 1

To visualize the effect of TseH on the cell wall, this video shows time-lapse fluorescence microscopy of E. coli cells expressing TseH (left), VgrG3 (middle) or TseL (right) effectors tagged to the periplasmic signal TAT. Cells were stained with 100 µg mL−1 of the PG-specific dye wheat germ agglutinin and spotted onto an agarose pad containing either 0.2% L-arabinose (TseH) or 1 mM IPTG (VgrG3 and TseL). Images were acquired every 20 sec during 25 min. Movie plays at a rate of 10 frames per second. Top row is a merge of phase and green (wheat germ agglutinin) channels and the bottom row shows green channel only. Scale bars, 5 µm. When expressing either TseH or the known cell-wall-targeting effector VgrG3, cells showed typical blebbing followed by PG sacculus deformation and cell lysis, whereas the effector TseL caused retraction and leaking of cytoplasmic content without obvious effect on PG. Video is representative of three independent replicates.

Source data

Source Data Fig. 1

Data and statistical source data used for Fig. 1.

Source Data Fig. 2

Data and statistical source data used for Fig. 2.

Source Data Fig. 3

Data and statistical source data used for Fig. 3.

Source Data Fig. 4

Data and statistical source data used for Fig. 4.

Source Data Fig. 5

Data and statistical source data used for Fig. 5.

Source Data Extended Data Fig. 1

Data and statistical source data used for Extended Data Fig. 1.

Source Data Extended Data Fig. 1

Uncropped western blots shown in Extended Data Fig. 1e.

Source Data Extended Data Fig. 3

Data and statistical source data used for Extended Data Fig. 3c,d. Please note that this is the same data as in Source Data for Fig. 2.

Source Data Extended Data Fig. 4

Data and statistical source data used for Extended Data Fig. 4.

Source Data Extended Data Fig. 5

Data and statistical source data used for Extended Data Fig. 5.

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Hersch, S.J., Watanabe, N., Stietz, M.S. et al. Envelope stress responses defend against type six secretion system attacks independently of immunity proteins. Nat Microbiol 5, 706–714 (2020). https://doi.org/10.1038/s41564-020-0672-6

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  • DOI: https://doi.org/10.1038/s41564-020-0672-6

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