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Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells

Abstract

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion–site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin–immunity pairs, including Tse8–Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing–based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.

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Fig. 1: Tse8–Tsi8 is a H1-T6SS toxin–immunity pair.
Fig. 2: Tse8 interacts with VgrG1a and does not require putative catalytic residue for toxicity.
Fig. 3: Tse8 targets the transamidosome.
Fig. 4: Tse8 impacts on protein synthesis in vivo.

Data availability

PAK genome NCBI number is LR657304, and in the ENA (European Nucleotide Archive) the accession code is ERS195106. The resulting sequences of the T6SS TraDIS assays are available from the ENA under study accession number ERS577921. Source data are provided with this paper.

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Acknowledgements

L.M.N. was supported by Medical Research Council (MRC) Grant MR/N023250/1 and a Marie Curie Fellowship (PIIF-GA-2013-625318). A.F. was supported by MRC Grants MR/K001930/1 and MR/N023250/1 and Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/N002539/1. R.C.D.F. and D.A.I.M. were supported by the MRC Career Development Award MR/M009505/1. D.A.-J. acknowledges support by the MINECO Contract CTQ2016-76941-R, Fundación Biofísica Bizkaia, the Basque Excellence Research Centre (BERC) program and IT709-13 of the Basque Government, and Fundación BBVA. M.A.S.-P. was supported by the MINECO under the “Juan de la Cierva Postdoctoral program” (position FJCI-2015-25725). Technical support from the CIC bioGUNE Metabolomics and Proteomics platforms are gratefully acknowledged.

Author information

Affiliations

Authors

Contributions

L.M.N. designed the overall experimental plan for the manuscript, performed the majority of the experiments presented and wrote the manuscript. A.K.C. performed all TraDIS sequencing and associated bioinformatic analyses. T.C. and R.C.D.F. performed pull-down experiments and bacterial growth assays. E.M. assisted with protein pull-down assays. M.A.S.-P. and D.A.-J. performed protein purification and MS enzymatic assays. D.A.-J. performed homology modelling and bioinformatic analyses; G.D. and J.P. contributed to project management and supported TraDIS sequencing and associated bioinformatic analyses. D.A.I.M. contributed to project management, designed experiments, performed protein purification and pull-down experiments, and contributed to the writing of the manuscript. A.F. contributed to project management, designed the overall experimental plan for the manuscript and contributed to writing the manuscript.

Corresponding authors

Correspondence to Despoina A. I. Mavridou or Alain Filloux.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 TraDIS library generation and sequencing workflow and predicted outcome of transposon insertions in tsi (immunity) genes in each library background.

a, En masse transposon (Tn) mutagenesis in T6SS active (PAKΔretS) or T6SS inactive (PAKΔretSΔH1) backgrounds was performed to generate pooled transposon mutant libraries of ~2 million mutants each. These libraries were then separately passaged overnight at high contact density and the genomic DNA from recovered mutants was harvested. This genomic DNA was then fragmented and adaptors ligated to each end prior to PCR enrichment for transposon-containing DNA fragments. The pooled DNA population was then subjected to TraDIS DNA sequencing. b, Artemis (http://www.sanger.ac.uk/science/tools/artemis - version 17/0.1) plot file showing distribution of transposon insertions (red and blue lines correspond to insertions mapped from either forward or reverse sequence reads) in immunity gene (tsi2 in this case) in the T6SS active library background (top panel - no insertions permitted) and in the T6SS inactive library background (right - insertions are permitted). The other H1-T6SS immunity genes detected, as well as the putative previously unidentified T6SS immunity genes (Table 1) had a similar distribution of transposon insertions in each library background as for tsi2. Panel (a) adapted from Barquist et al., (2013)56.

Extended Data Fig. 2 Genomic context of putative toxin-immunity pairs identified in TraDIS screen.

Putative toxin and immunity pairs from Table 1 are in orange with surrounding genes in blue. Genes corresponding to PAO1 ORF numbers. Base pairs covering the region are marked below each gene sequence.

Extended Data Fig. 3 Prey killing is mediated by Tse8 and effects can be complemented by expressing Tse8 or Tsei8 in trans.

a, In the absence of Tse8 (PAKΔretSΔtse8) or the H1-T6SS (PAKΔretSΔH1) there is no reduction in recovered recipient (PAKΔretSΔtsei8) as occurs when the donor has a fully active T6SS (PAKΔretS). b-c, The PAKΔretSΔtsei8 (b) or PAKΔretSΔtse8 (c) mutation can be complemented in trans. Competition assays were performed with donors PAKΔretS or PAKΔretSΔH1 and recipient PAKΔretSΔtsei8 with either empty pBBR1MCS5 or the complementation vector pBBR1:tsei8 (b) or recipient PAKΔretSΔtsei8 with either empty pBBR1MCS4 or the complementation vector pBBR1:tse8 (c). Statistical analyses: a, Mean CFUs/mL ± SEM of recipient cells in competition/alone are represented from represented from three independent replicates performed in triplicate (n=3). Two-tailed student’s t-test, * p<0.05 for PAKΔretS [pBBR1-MCS4] vs recipient compared to PAKΔretSΔtse8 [pBBR1-MCS4] vs recipient, PAKΔretSΔtse8 [pBBR1-MCS4] vs recipient compared to PAKΔretS [pBBR1:tse8] or PAKΔretSΔtse8 [pBBR1:tse8] vs recipient. b, Mean CFUs/mL ± SEM of recipient cells in competition/alone are represented from represented from five independent replicates performed in triplicate (n=5). Two-tailed student’s t-test, ** p<0.005 compared to PAKΔretS donor vs recipient PAKΔretSΔtsei8 [pBBR1-MCS5] compared separately to the other datasets; ns between recovered CFUs/mL for recipient PAKΔretSΔtsei8 [pMMB-MCS5] vs PAKΔretSΔH1 (p=0.51) and recipient PAKΔretSΔtsei8 [pMMB:tsei8] vs PAKΔretS (p=0.61). c, Mean CFUs/mL ± SEM of recipient cells in competition/alone are represented from represented from three independent replicates performed in triplicate (n=3). Two-tailed student’s t-test, ** p<0.005 for PAKΔretS donor vs recipient compared separately to the other datasets.

Source data

Extended Data Fig. 4 Sequence alignment of Tse8 with predicted homologs of known 3D structure.

Amino acid sequences from P. aeruginosa Tse8 (Pa.Tse8), Stenotrophomonas maltophilia peptide amidase (Sm.Pam), Staphylococcus aureus Gln-tRNA(Gln) amidotransferase subunit A (Sa.GatA), P. aeruginosa Asn-tRNA(Asn) transamidosome subunit A (Pa.GatA), Flavobacterium sp. 6-aminohexanoate cyclic dimer hydrolase NylA (Fsp.NylA), Bradyrhizobium japonicum malonamidase E2 (Bj.MAE2), Pseudomonas sp. allophanate hydrolase (Psp.AtzF) and Bacterium csbl00001 Aryl Acylamidase (9BACT.AAA) were aligned. Residues are colour-coded depending on the percentage of equivalences; white letter in red background for residue 100 % conserved, red letter in white background for residue with physical-chemical properties conserved. The secondary structure elements found in the 3D structure of Sm.PAM are represented above the alignment (black arrows correspond to β-sheets and curly lines to α-helices). The conserved Ser-Ser-Lys catalytic triad is indicated below the alignment by black circles. The AS signature sequence is indicated below the alignment by a dotted line. Regions that protrude out of the core AS domain are numbered below the alignment. Residues found to interact with substrates/substrate analogues, products or inhibitors are indicated with black triangles below the alignment (analysis was carried out for crystal structures with the following PDB codes: 1M21 (Sm.Pam), 1O9O (Bj.MAE2), 4CP8 (Psp.AtzF) and 4YJI (9BACT.AAA)). Alignment was generated using MUSCLE57 and graphical representation was performed with ESPript 358.

Extended Data Fig. 5 Recombinant production of Tse8.

a, Amino acid sequence of Pseudomonas aeruginosa GST-TEV-Tse8 construct. The recombinant Tse8 construct contains a fused glutathione S-transferase (GST) tag (grey colour), a S15 tag (dashed line), a poly-histidine tag (smooth line) and the optimal Tobacco Etch Virus protease (TEV) cleavage site (ENLYFQG) (dotted line) at the N-terminus of Tse8 (in blue letters). b, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified Tse8. Lane 1, molecular weight marker; Lane 2, sample before cleaving with TEV; Lanes 3-4, sample after incubation with TEV; Lane 5, Tse8 without tags (4-20% gel (ExpressPlus™ PAGE Gel, GenScript). c, Deconvoluted electrospray ionization mass spectrometry (ESI-MS) chromatogram of purified Tse8 after TEV cleavage (the experimentally determined molecular weight corresponds to the expected molecular weight of 60,564 Da). Experiments described in (b) and (c) were performed in one biological replicate.

Extended Data Fig. 6 Tse8 is not active on a substrate of the amidase Pam.

MS analysis of Tse8 (a) or Pam (b) enzymatic assay using epinicedin-1 as substrate. The antimicrobial peptide epinecidin-1, as well as sermorelin, have amidated C termini. The latter has previously been used to measure the amidase activity of Pam from S. maltophilia55. In both (a) and (b): Sequence covered by the fragments obtained after fragmentation of epinecidin-1 in the MS is indicated above the fragmentation spectra for epinecidin-1. Signals corresponding to the amidated (a) or deamidated (b) form of epinecidin-1 are shown in the spectral plot with red ions belonging to the b series of fragments, blue ions to the y series, and green ions to parental forms of the peptide. See Extended Data Fig. 7 for correspondence between the observed fragments and their theoretical masses.

Extended Data Fig. 7 Theoretical masses for the fragments detected during MS analysis of the amidase reactions presented in Extended Data Fig. 6.

In both (a) and (b): Correspondence between the observed fragments and their theoretical masses. Tables correspond to unaltered ion series at +1 and +2 charge states (left), ion series after neutral losses at +1 and +2 charge states (ammonia loss, centre) and parental ion masses at +2 charge state with or without diverse neutral losses (right). Red ions belong to the b series of fragments, blue ions to the y series, and green ions to parental forms of the peptide. See Extended Data Fig. 6 for corresponding spectral plots.

Extended Data Fig. 8 Tse8 is not active on a substrate of GatA.

a, Amidase reaction catalysed by GatA and b, MS analysis of Tse8 enzymatic assay using glutamine as substrate. Signal of the expected product (glutamate) was also found as a contaminant in the blank and product stock and therefore subtracted from the reaction incubation. The graph on the left shows relative differences (%Δ) of glutamate and glutamine in reaction incubation and blank samples. The green-shaded area indicates the zone in which the observed differences could indicate enzymatic reaction. (%ΔProduct = 100 * ([product in incubation] – [product in blank]/ [product in incubation]; %ΔSubstrate = 100 * ([substrate in incubation] – [substrate in blank]/ [substrate in incubation]). The graph on the right shows the ratios between substrate and product (Rsp) in the blank (red) and reaction (Tse8) incubation (blue) samples. The product signal (contaminant) was ca. 100 times lower than that of the substrate. (Rsp = (Signal substrate / Signal product)). c, Glutaminase assays of lysates of E. coli cells expressing GatA, Tse8 or empty vector demonstrate that Tse8 does not have the same substrate (L-glutamine) as GatA as measured by relative NADPH levels/(CFU/mLx108) and here normalized to empty vector (EV) mean. Mean ± SEM of six biological replicates performed in triplicate (n=6). Two-tailed student’s t-test, ** p<0.005 for empty vector compared to pET41a:gatA; ns for empty vector compared to pET41A:tse8 (p=0.621).

Source data

Extended Data Fig. 9 Tse8 is structurally similar to GatA of the transamidosome complex.

a, Structure of the P. aeruginosa GatCAB transamidosome-Asp-tRNA structure (PDB: 4WJ3). b, Top panel: Tse8 3D homology model generated using GatA from S. aureus (from PDB: 2F2A) as template overlaid with the A subunit of the solved GatCAB transamidosome-AspS-tRNA structure from P. aeruginosa (PDB: 4WJ3). The reaction centre with covalently bound glutamine substrate is boxed. Bottom panel: Close-up view of the reaction centre of S. aureus GatA (left) with glutamine (green) substrate bound and of a superposition of S. aureus GatA and the 3D homology model of P. aeruginosa Tse8 (right) showing the predicted conservation of the Ser-cisSer-Lys catalytic triad and predicted divergent substrate binding residues in Tse8 compared to GatA.

Extended Data Fig. 10 Volcano plot showing the spread of changes in abundance of TraDIS mutants for each P. aeruginosa gene during T6SS active compared to inactive conditions.

Each black dot represents the comparative fold change of insertions for each gene. Red lines show the cut-off criteria of 5% false discovery rate (horizontal) and a log2 fold change (Log2FC) of 2 (vertical). The corresponding Log2 Fold-Change values on the x-axis for each gene are reported Supplementary Table 1. A -Log2 transformation has been applied to the corresponding Q-values for each gene as reported in Supplementary Table 1. Immunity genes and putative immunity genes (as shown in Table 1) are shown in blue.

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Legend for Supplementary Table 1 and Tables 2–4.

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Supplementary Table 1

Contains the full TraDIS data set (Tab 1), data above our threshold cut-off (-logFC > 2 q< 0.05) (Tab 2) and data from Tab 2 limited to genes <600 bp (Tab 3). See ‘Generation of TraDIS sequencing libraries, sequencing and downstream analysis’ in Methods for details on analyses. P values were corrected for multiple testing using the Benjamini–Hochberg method, and genes with a corrected P value (Q value) of <0.05 (5% false discovery rate) and an absolute log2(fold change) (log2FC) of >2 were considered significant.

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Nolan, L.M., Cain, A.K., Clamens, T. et al. Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells. Nat Microbiol 6, 1199–1210 (2021). https://doi.org/10.1038/s41564-021-00950-8

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