Identification of Divergent Type VI Secretion Effectors Using a Conserved Chaperone Domain

Identification of divergent type VI secretion effectors using a conserved chaperone domain

Xiaoye Lianga,b,c, Richard Moorea,b,c, Mike Wiltona,b,c, Megan J. Q. Wonga,b,c, Linh Lama,b,c, and Tao G. Donga,b,c,1

aEcosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada T2N 4Z6; bSnyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada T2N 4Z6; and cBiochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada T2N 4Z6

Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved June 5, 2015 (received for review March 18, 2015)

The type VI secretion system (T6SS) is a lethal weapon used by membrane of eukaryotic cells (18, 21, 22) and the cell wall, mem- many bacteria to kill eukaryotic predators or prokaryotic compet- brane, and DNA of bacterial cells (3, 18–20, 23, 24). Each anti- itors. Killing by the T6SS results from repetitive delivery of toxic bacterial effector coexists with an antagonistic immunity protein effectors. Despite their importance in dictating bacterial fitness, that confers protection during T6SS-mediated attacks between systematic prediction of T6SS effectors remains challenging due to sister cells (3, 18, 24). Interestingly, T6SS-mediated lethal attacks high effector diversity and the absence of a conserved signature induce the generation of reactive oxygen species in the prey cells sequence. Here, we report a class of T6SS effector chaperone (TEC) (25), similar to cells treated with antibiotics (26, 27). – proteins that are required for effector delivery through binding to For non-VgrG/PAAR related effectors, their translocation VgrG and effector proteins. The TEC proteins share a highly con- requires either binding to the inner tube Hcp proteins as chap- served domain (DUF4123) and are genetically encoded upstream erones or binding to the tip VgrG proteins (2, 14, 28). T6SS- dependent effectors can be experimentally identified by com- of their cognate effector genes. Using the conserved TEC domain – sequence, we identified a large family of TEC genes coupled to paring the secretomes of WT and T6SS mutants (3, 29 31) and by screening for T6SS-encoded immunity proteins (18). Because putative T6SS effectors in Gram-negative bacteria. We validated known effectors lack a common secretion signal, bioinformatic this approach by verifying a predicted effector TseC in Aeromonas hydrophila identification of T6SS effectors is challenging. A heuristic ap- . We show that TseC is a T6SS-secreted antibacterial proach based on the physical properties of effectors has been

tsiC MICROBIOLOGY effector and that the downstream gene encodes the cognate used to identify a superfamily of peptidoglycan-degrading ef- immunity protein. Further, we demonstrate that TseC secretion fectors in bacteria (32). A recent study identified a common requires its cognate TEC protein and an associated VgrG protein. N-terminal motif in a number of T6SS effectors (31). However, this Distinct from previous effector-dependent bioinformatic analyses, motif does not exist in the T6SS effector TseL in V. cholerae (18). our approach using the conserved TEC domain will facilitate the In this study, we report that VC1417, the gene upstream of tseL, discovery and functional characterization of new T6SS effectors in encodes a protein with a highly conserved domain, DUF4123. Gram-negative bacteria. We show that VC1417 is required for TseL delivery and interacts with VgrG1 (VC1416) and TseL. Because of the genetic linkage interspecies interaction | colicin | antitoxin | toxin | protein secretion of VC1417 and TseL and its importance for TseL secretion, we postulated that genes encoding the conserved DUF4123 domain rotein secretion systems play a pivotal role in bacterial in- proteins are generally located upstream of genes encoding pu- Pterspecies interaction and virulence (1, 2). Of the known tative T6SS effectors. Using the conserved domain sequence, we secretion systems in Gram-negative bacteria, the type VI secre- bioinformatically predicted a large family of effector proteins with tion system (T6SS) enables bacteria to compete with both eukary- diverse functions in Gram-negative bacteria. We validated our otic and prokaryotic species through delivery of toxic effectors (2– prediction by the identification and characterization of a new 4). The T6SS is a multicomponent nanomachine analogous to the contractile bacteriophage tail (5). First characterized in Significance Vibrio cholerae (6) and Pseudomonas aeruginosa (7), the T6SS has now been identified in ∼25% of Gram-negative bacteria, How different microbial species compete for specific niches is including many important pathogens (2, 8), and has been im- not fully understood. Here, we focus on the type VI secretion plicated as a critical factor in niche competition (9–11). system (T6SS), a specialized protein delivery system, which The T6SS structure is composed of an Hcp inner tube, a many Gram-negative bacteria use to kill eukaryotic and pro- VipAB outer sheath that wraps around the Hcp tube, a tip karyotic competitors by translocating toxic protein molecules complex consisting of VgrG and PAAR proteins, and a mem- to target cells. Identification of effectors is required for un- brane-bound baseplate (2, 4, 12). Sheath contraction drives the derstanding the pivotal role that the T6SS plays in dictating inner Hcp tube and the tip proteins, VgrG and PAAR, outward interbacterial and bacterial–host dynamics. In this study, we into the environment and neighboring cells (13, 14). The con- describe a new approach to systematically identify T6SS ef- tracted sheath is then dissembled by an ATPase ClpV and fectors. We also demonstrate that secretion of effectors re- recycled for another T6SS assembly and contraction event (12, quires interaction with a set of cognate effector-binding 15, 16). Two essential T6SS baseplate components, VasF and chaperone proteins that we define in this study, providing VasK, are homologous to the DotU and IcmF proteins of the important insights for understanding the mechanism of T6SS type IV secretion system (T4SS) in Legionella pneumophila (17). effector delivery. Bacteria often possess multiple copies of VgrG and PAAR genes that form the tip of T6SS, and deletion of VgrG and Author contributions: X.L. and T.G.D. designed research; X.L., R.M., and T.G.D. performed PAAR genes abolishes T6SS secretion (14). Some VgrG and research; T.G.D. contributed new reagents/analytic tools; X.L., R.M., M.W., M.J.Q.W., L.L., PAAR proteins carry functional extension domains and thus act and T.G.D. analyzed data; and T.G.D. wrote the paper. as secreted T6SS effectors, as exemplified by the VgrG1 actin The authors declare no conflict of interest. cross-linking domain (6), VgrG3 lysozyme domain in V. cholerae This article is a PNAS Direct Submission. (18, 19), and the nuclease domain of the PAAR protein RhsA in 1To whom correspondence should be addressed. Email: [email protected]. Dickeya dadantii (20). Known T6SS effectors can target a num- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ber of essential cellular components, including the actin and 1073/pnas.1505317112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1505317112 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 secreted effector TseC and its antagonistic immunity protein used a bacterial two-hybrid assay based on the functional com- TsiC in Aeromonas hydrophila SSU. Our results demonstrate a plementation of the two T18 and T25 fragments of Bordetella new effective approach to identify T6SS effectors with highly pertussis adenylate cyclase (34). Protein interaction functionally divergent sequences. reconstitutes the activity of adenylate cyclase that subsequently + results in a LacZ phenotype on LB supplemented with X-gal. Results Because VgrG1 carries a large C-terminal actin–cross-linking Secretion of TseL Requires VC1417 and VgrG1 in V. cholerae. TseL domain that can be swapped by beta-lactamase without affecting (VC1418) is located in the V. cholerae hcp1 operon consisting of secretion (35), we reasoned that the C-terminal extension do- seven genes (Fig. 1A), three of which (VC1417, VC1420, and main is not required for delivery and thus expressed only the highly conserved N-terminal sequence (1-638aa) of VgrG1 for VC1421) encode proteins with unknown functions. We first + tested whether any of these three genes were required for TseL testing protein–protein interaction. We found LacZ pheno- secretion. By comparing WT and a mutant lacking VC1417 to types when VC1417 was coexpressed with TseL or VgrG1, VC1421 (33), we found that TseL cannot be secreted in the suggesting direct interaction (Fig. 1F). In contrast, the nega- B tive control VasH, a DNA-binding sigma54-dependent regu- mutant (Fig. 1 ), indicating that at least one of the three genes, − VC1417, VC1420, and VC1421, is required for TseL secretion. lator (36–38), exhibited LacZ when coexpressed with the We reasoned that, if complementation of a gene can restore other proteins tested. Using coimmunoprecipitation assays, TseL secretion in the VC1417-21 mutant that lacks the immunity weconfirmedtheinteractionofVC1417withVgrG1andTseL gene VC1419, it would be highly toxic when coexpressed with (Fig. 1G). TseL due to sister cell-to-sister cell delivery of TseL. Indeed, we found that coexpression of VC1417 and TseL reduced cell sur- VC1417 Belongs to a Large Family of Proteins with a Conserved vival by 103-fold whereas expressing VC1417 or TseL alone had Domain. By searching the protein sequence of VC1417 in the little effect on cell viability (Fig. 1C), indicating that VC1417 is Pfam protein database (39), we found that VC1417 carries a required for TseL secretion. Interestingly, VC1417 itself is not a conserved domain DUF4123. DUF4123 was found in 818 pro- secreted substrate of T6SS (Fig. 1D). Expression of VC1420 or tein sequences in 344 bacterial species, 342 of which belong VC1421 did not restore TseL secretion. to Proteobacteria, including Gammaproteobacteria (69%) and We then tested whether the vgrG1 gene, upstream of VC1417, Betaproteobacteria (26%) (Fig. 2A and Dataset S1). Although is involved in TseL secretion in V. cholerae. Using the VC1417-21 DUF4123 is the only domain in the majority of these proteins, a mutant as a prey, we found that WT V. cholerae killed the prey few proteins carry an additional forkhead-associated (FHA) efficiently whereas the vgrG2 mutant could not kill (Fig. 1E). domain that is often involved in regulatory functions through This observation is consistent with previous reports that VgrG2 phosphorylation. Interestingly, the Fha1 protein in P. aeruginosa is essential for T6SS secretion (18, 33). Interestingly, whereas the is required for activating the T6SS cluster 1 (40). In addition, vgrG3 mutant exhibited impaired killing, killing was completely over 90% of DUF4123-encoding bacterial genomes also carry abolished in the vgrG1 mutant (Fig. 1E). As a control, the vgrG1 hallmark T6SS proteins, VipA (the outer sheath), and Hcp (the mutant efficiently killed Escherichia coli as a prey (Fig. S1), inner tube), indicating a strong association between the presence consistent with a previous report (33). These results indicate that of DUF4123 and T6SS genes (Fig. 2B). VgrG1 is not essential for T6SS activity but is required for de- The genome of V. cholerae encodes another DUF4123 domain livery of TseL. We previously found that VgrG3 interacts with protein, VasW, which is known to be required for secretion of TseL in V. cholerae but not in E. coli (18). Because VgrG pro- its downstream effector, VasX (23). Because the DUF4123 teins likely form a heterotrimer in V. cholerae (21, 33), our results domain proteins are widely distributed in Gram-negative bac- suggest that the previously reported interaction between VgrG3 teria, we reasoned that we could use this conserved domain as a and TseL is likely through VgrG1. signal to find highly divergent T6SS effectors. Two previously characterized effectors in V. cholerae, TseL and VasX, share Interaction Between TseL, VC1417, and VgrG1. The requirement of little sequence similarity, but both have the DUF4123 domain- VC1417 and VgrG1 for TseL delivery suggests that these pro- containing genes upstream, validating this method as a potential teins may interact with one another. To test this hypothesis, we strategy for T6SS effector identification.

Fig. 1. VC1417 is important for TseL secretion in V. cholerae.(A) Operon structure of the VC1415- VC1421 region. Three of the seven genes, highlighted in black, have unknown functions. (B) Immunoblot- ting analysis of TseL::3V5 secretion as detected in the cytosolic (Cell) and supernatant (Sec) fractions. RpoB is an abundant cytosolic protein and is used as a control for cell lysis. (C) VC1417 is required for delivery of TseL. The VC1417-21 mutant was complemented with a plasmid-borne VC1417 or TseL alone or two plas- mids carrying both VC1417 and TseL. Survival of the VC1417-21 mutant was tested by plating strains on LB medium containing 0.1% arabinose. Restoration of TseL delivery by VC1417 resulted in toxic delivery of TseL between sister cells, thereby leading to cell death. Mean values and SEs of triplicate samples are indicated. (D) VC1417 is not secreted by T6SS. VC1417::3V5 was overexpressed using a pBAD arabi- nose-inducible vector, and secretion was tested by immunoblotting. Wild type, V. cholerae T6SS active strain V52; ΔT6SS, the vasK deletion mutant of V52. (E) Effects of VgrG proteins on TseL delivery. Prey cell is the VC1417-21 mutant lacking the cognate immunity gene tsiV1 to TseL. Killer cells are WT V52 and its derivative vgrG and vasK mutants as indicated. (F) Bacterial two-hybrid analysis of VC1417 interaction with VgrG1 and TseL. VasH is a DNA-binding sigma54- dependent regulator and was used as a control. (G) Coimmunoprecipitation of VC1417 with VgrG1 and TseL. Cell lysates containing VC1417-FLAG alone or together with VgrG1-6His or TseL-6His were incubated with anti-His magnetic beads, and bound proteins were analyzed by Western blot using an anti-FLAG monoclonal antibody.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1505317112 Liang et al. Downloaded by guest on October 1, 2021 complexes are important virulence factors in many bacterial pathogens, including Photorhabdus luminescens and Yersinia pestis, which target insects and mammalian cells (46, 47). We thus named ORF2402 TseC for its colicin domain and ORF0928 TseI for its potential insecticidal activity. We expanded our analysis to 43 representative bacterial species with completely annotated genomes that encode the DUF4123 proteins (Dataset S2). For genes immediately upstream of the 133 DUF4123 genes analyzed, 70% possess an upstream vgrG and 7.5% an upstream PAAR (Dataset S2). For DUF4123 down- stream genes, whereas the majority encode unknown functions, genes with known/predicted functions encode putative TC-toxin, Fig. 2. Distribution of the DUF4123 domain in bacterial species. (A) Distri- lipase, nuclease, and hydrolase. bution of DUF4123 in Proteobacteria. DUF4123 species were retrieved from the Pfam database. (B) Comparison of bacterial genomes possessing the TseC Is a T6SS-Dependent Antibacterial Effector in A. hydrophila DUF4123 domain proteins and the T6SS hallmark proteins VipA and Hcp. SSU. To functionally validate our predictive method, we used A. hydrophila SSU as a model. The T6SS of SSU is known to target eukaryotic cells (42, 48), but T6SS-mediated antibacterial As a proof of principle, we first examined DUF4123 and down- activities have not been demonstrated. To assess the function stream genes in a number of known T6SS-active bacteria, including of the T6SS in A. hydrophila, we constructed a T6SS-null mu- P. aeruginosa (41), Agrobacterium tumefaciens (10), D. dadantii (20), tant lacking the vasK gene essential for T6SS functions (42). and Aeromonas hydrophila (42). Indeed, genes encoding the Using a bacterial killing assay (18), we found that WT SSU DUF4123 domain are found upstream of genes encoding known killed E. coli by 10,000-fold in comparison with the vasK mutant T6SS effectors. Notably, DUF4123 genes are also often located (Fig. 4A), indicating that the T6SS of SSU is highly effective in together, with at least of one of the genes encoding T6SS secreted interbacterial competition. proteins Hcp, VgrG, or PAAR (Fig. 3). To test whether the predicted effector TseC is secreted by Next, we tested whether the DUF4123 domain can predict A. hydrophila T6SS (Fig. 3), we expressed an epitope-tagged unknown T6SS effectors. P. aeruginosa PA14 carries four TseC in the WT and the vasK mutant. Western blot analysis

DUF4123 proteins, one of which is located upstream of a known showed that TseC was secreted only by the WT but not the vasK MICROBIOLOGY T6SS effector RhsP2 (41). The other three DUF4123 genes are mutant (Fig. 4B). TseC is predicted to carry a colicin domain located immediately downstream of genes encoding VgrG or (Fig. 4C). Because colicins attack E. coli through binding to cell PAAR proteins (Fig. 3). Using a structural prediction program, membranes (49), we determined TseC antibacterial toxicity by HHpred (43), we found that the three genes downstream of each expressing the colicin domain in the periplasm of E. coli using a of the DUF4123 genes encode a hydrolase, a endonuclease, twin-arginine secretion signal (50, 51). Periplasmic expression of and a colicin, respectively, suggesting these gene products are the colicin domain reduced the survival of E. coli to 1% after T6SS effectors. There are two DUF4123 domain proteins in induction (Fig. 4D), indicating that A. hydrophila TseC is po- A. hydrophila SSU (Fig. 3). Sequence analyses using HHpred (43) tently antibacterial. and Phyre2 (44, 45) show that the ORF2402 carries a putative Because previously characterized T6SS-dependent toxic ef- colicin domain (HHpred probability 21% and Phyre2 confidence fectors coexist with antagonistic immunity proteins that are 78%) and that the ORF0928 carries a TC toxin domain (HHpred encoded by downstream genes (18, 24), we predicted that the probability 100% and Phyre2 confidence 100%). The TC toxin gene downstream of tseC is the cognate immunity gene, hereafter

Fig. 3. Known and predicted DUF4123-associated effectors. DUF4123-containing genes were retrieved from the Pfam database. Protein functions were predicted using the HHpred program. Colors indicate genes encoding functionally similar proteins from a diverse set of Gram-negative bacteria.

Liang et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Fig. 4. Identification of an effector-immunity pair TseC-TsiC in SSU. (A) T6SS-dependent killing of E. coli by SSU. WT SSU and its T6SS null mutant vasK were mixed with prey E. coli at a 10:1 ratio and spotted on LB medium. Survival of E. coli was enumerated by serial dilution. (B) T6SS-dependent secretion of TseC. TseC carrying a 3V5 tag was detected in whole cell lysate or the supernatant (secreted) by immunoblotting analysis using an anti-V5 antiserum. (C) Operon structure of SSU tseC-tsiC. TseC carries a predicted colicin domain. (D) Expression of the SSU TseC colicin domain (768–891) is toxic in E. coli. The colicin domain was cloned into a pBAD vector under an arabinose-inducible promoter. A twin-arginine translocation (Tat) signal sequence was added to the N-terminalto deliver the colicin domain to the periplasm. (E) TsiC is the cognate immunity protein to TseC. The SSU deletion mutant of tseC tsiC was sensitive to T6SS killing whereas complementation with TsiC restores protection. Killer cells are WT SSU and its vasK mutant. Prey cells are the tseC tsiC double mutant and the tseC tsiC mutant complemented with a plasmid-borne tsiC.(F) ORF2403 and VgrG1 (ORF2404) are required for killing the tseC tsiC mutant but not E. coli. Killer cells are WT SSU and its mutant derivatives as indicated. Prey cells are the tseC tsiC double mutant of SSU and E. coli K-12 MG1655. (G) Bacterial two-hybrid assay of the interaction of ORF2403 with VgrG1(ORF2404) and TseC. VasH, a DNA-binding regulator in V. cholerae, is included as a negative control. (H) Coimmu- noprecipitation analysis of the interaction of ORF2403 with VgrG1(ORF2404) and TseC. Cell lysates containing ORF2403-6His alone or with VgrG1-3V5 or TseC-3V5 were incubated with magnetic beads conjugated with a monoclonal anti-V5 antibody. Bound proteins were detected using a monoclonal antibody to 6His.

referred to as tsiC. If TsiC is the immunity protein to TseC, the humans and plants. Despite their importance, the identification tsiC mutant would be susceptible to WT T6SS-mediated killing and assignment of enzymatic function to T6SS effectors still by delivery of TseC. We tested this hypothesis by constructing a remains challenging. Comparative analysis of effector sequences double knockout mutant lacking both tseC and tsiC. Indeed, the from different species could be used to identify potential ho- tseC tsiC mutant was efficiently killed by 104-fold when exposed mologs. However, systematic identification of effectors using to WT SSU, and complementation with a plasmid-borne tsiC bioinformatics is difficult because known T6SS effectors are fully protected the tseC tsiC mutant from killing (Fig. 4E), in- highly diverse in sequence and function. Although previous dicating that TsiC is the cognate immunity protein to TseC. studies have successfully identified a number of effectors based on the physical characteristics of known effectors (32) and an Delivery of TseC Requires VgrG1 and DUF4123 Protein ORF2403. Up- N-terminal sequence marker (31), respectively, neither method stream of SSU tseC are vgrG1 (ORF2404) (48) and the DUF4123 could identify TseL in V. cholerae. gene ORF2403. We postulated that the secretion of TseC re- In this study, instead of relying on the diverse effector se- quires VgrG1 and ORF2403 in SSU. To test this hypothesis, we quences, we demonstrate an effective approach of using a con- constructed deletion mutants of vgrG1 and ORF2403 and tested served domain (DUF4123) to identify the associated downstream their effects on killing the tseC tsiC double mutant. Neither mu- effectors. Our results show that DUF4123 proteins directly in- tant could kill the tseC tsiC double mutant (Fig. 4F). In contrast, teract with the cognate VgrG and effector proteins and play an E. coli was efficiently killed by these mutants, indicating that essential role in effector delivery but that DUF4123 proteins are ORF2403 and VgrG1 are not required for T6SS functions. Given not secreted or required for effector activities. DUF4123 may thus function similarly to the chaperone proteins of T4 phage, gp38 the dependency of TseC delivery on ORF2403 and VgrG1, we (53, 54) and gp63 (55, 56), which are important for tail fiber as- sought to determine whether these proteins interact. Using the sembly and attachment but are not components of the mature bacterial two-hybrid assay (Fig. 4G) and coimmunoprecipitation phage particle (57). In addition, the secretion of many effectors of (Fig. 4H), we identified the interaction of ORF2403 with VgrG1 the type 3 secretion (TTS) system is dependent on specific in- and TseC. In addition, the tseC mutant could still kill E. coli, teraction with cognate chaperone proteins that are present in the suggesting the existence of other T6SS-dependent antibacterial cytosol but not secreted (58, 59). Interestingly, TTS chaperone effectors in SSU (Fig. S2). proteins generally have low molecular weight and acidic isoelectric point (pI <5), and the chaperone genes are often found next to Discussion the genes encoding cognate effectors (58, 59). We found that Since the discovery of the T6SS in V. cholerae and P. aeruginosa, DUF4123 proteins also exhibit low pI values (∼5) and that considerable effort has been made toward understanding the the domain has several highly conserved residues (Fig. S3). Be- delivery mechanism and the physiological functions (2, 4, 14, cause of the abovementioned characteristics of DUF4123 proteins, 52). Previous research highlights that numerous human path- we postulate that DUF4123 proteins function as T6SS effector ogens use the T6SS to deliver toxic effectors to their bacterial chaperones (TECs) and thus name the DUF4123 domain TEC, competitors or eukaryotic hosts (2, 4). Recent reports on T6SS the VC1417 protein TecL, and the SSU2403 protein TecC. function in the Bacteroidetes (11) and Agrobacterium (10) further TEC genes are widely distributed in Proteobacteria and are underline the importance of T6SSindictatingbacterialdy- largely located together with an upstream VgrG/PAAR gene. namics in complex communities, such as the microbiota in We predicted that downstream of TEC genes are genes encoding

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1505317112 Liang et al. Downloaded by guest on October 1, 2021 candidate T6SS effectors. Using the TEC sequence, we validated recombination (60, 61). Gene expression vectors were constructed as pre- our analyses by identifying known effectors, including TseL viously described (38). All constructs were verified by sequencing. Primers for and VasX in V. cholerae, that share few common features in cloning and mutant construction are listed in Dataset S3. sequence, function, and structure. We also confirmed a new T6SS-dependent effector-immunity pair, TseC-TsiC, in the Western Blotting Analysis. Protein samples were loaded on a precast 4–12% A. hydrophila SSU strain. SDS/PAGE gel (Life Technologies), run at 180 V for 40 min and transferred Although the exact mechanism for T6SS protein export is not to a PVDF membrane (Millipore) by electrophoresis. The membrane was fully understood, there are two predicted models. The first blocked with 5% (wt/vol) nonfat milk in TBST buffer (50 mM Tris, 150 mM model requires that effectors bind to the inner surface of the NaCl, 0.05% Tween 20, pH 7.6) for 1 h at room temperature, incubated with primary antibodies at 4 °C overnight, washed three times in TBST buffer, and ring-like Hcp hexamers (52) whereas the second, termed multi- incubated with an HRP-conjugated secondary antibody (Cell Signaling ple effector translocation VgrG (MERV), involves binding of Technology) for 1 h followed by detection using the ECL solution (Bio-Rad) effectors to the tip VgrG and PAAR proteins (2, 14, 18). The and a ChemiDoc MP system (Bio-Rad). The monoclonal antibodies to epitope limited inner space of the Hcp hexameric ring likely poses a tags, anti-V5, anti-FLAG, and anti-6xHIS were purchased from Sigma Aldrich. physical restraint on the size of effectors relying on binding to The monoclonal antibody to RpoB, the beta subunit of RNA polymerase, was Hcp as chaperones for delivery (4). In the MERV model, binding purchased from NeoClone and used as a loading control for Western blot to the tip proteins renders more flexibility to accommodate ef- analysis as previously described (62). fectors that differ greatly in size and sequence (2, 14). Indeed, a

number of effectors with diverse functions have been reported to Protein Secretion Assay. Exponential phase cultures (OD600 = 0.5) grown in LB require VgrG and PAAR proteins for delivery (14, 18, 41). Our were induced by adding 0.1% L-arabinose for 1 h. One-milliliter culture was results on the VgrG-dependent secretion of TseL in V. cholerae collected by centrifugation twice at 20,000 × g for 2 min and then filtered and TseC in A. hydrophila further support the MERV model. through a 0.2-μm filter. The filtered supernatant was combined with 200 μL Notably, many bacterial species possess multiple TEC pro- of 100% ice-cold TCA solution, placed on ice for 2 h, and centrifuged at teins. For example, A. hydrophila SSU has two TEC proteins 15,000 × g for 30 min at 4 °C. The pellet was washed with 1 mL of 100% (Fig. 3). These two TEC proteins cannot functionally comple- acetone by centrifugation at 20,000 × g for 5 min, air-dried and mixed with ment each other, as evidenced by the loss of killing resulting 30 μL of SDS-loading dye, followed by SDS/PAGE and Western blot analyses from deletion in tecC (ORF2403) (Fig. 4F). Because TEC pro- as described above. teins interact with both conserved VgrG proteins and divergent effectors, we propose that the conserved TEC domain is re- Bacterial Cell-Killing Assay. The killing assay was performed as previously sponsible for binding to VgrG/PAAR whereas each TEC protein described (33). Briefly, cultures were mixed together at a ratio of 10:1 has acquired specific sequences to accommodate binding to its (predator to prey), spotted on LB medium for 3 h at 37 °C, and then resus- MICROBIOLOGY pended in 1 mL of LB. Survival of prey cells was quantified by serial dilution partner effector. For T6SS-mediated delivery of a given VgrG- in LB and plating on selective medium. binding effector, multiple binding events likely occur in a tem- poral order that includes effector binding to the cognate VgrG, Bacterial Two-Hybrid Assay. The two-hybrid assay was performed as described to the TEC protein, and to the immunity protein (if the immu- (34, 63). Plasmid vectors carrying the indicated T18 and T25 constructs were nity protein is present in the cytosol). Because TEC proteins and transformed to BTH101 (cya-99). Individual colonies were grown in LB for 3 h immunity proteins are not secreted, the separation of effectors and then patched on LB medium supplemented with Amp, Kan, X-Gal, and from the cognate TEC and immunity proteins probably takes 0.5 mM IPTG. Plates were incubated at room temperature for at least 48 h. place before binding to VgrG for delivery. It is possible that TEC proteins coordinate the process of effector loading to the VgrG/ Coimmunoprecipitation Assay. Genes were cloned into pETDuet-1 and PAAR spike to prevent premature binding of effectors with pACYCDuet-1 vectors for expression. E. coli BL21DE3 carrying different gene VgrG. The formation of T6SS spike might expose the effector- expression vectors was grown in 50 mL of LB culture to exponential phase

binding site of VgrG that attracts effectors and displaces TEC OD600 = 0.5, and induced by 1mM IPTG for 3 h at 37 °C. Cells were collected by proteins. Because TEC proteins can bind to both effectors and centrifugation at 4,000 × g for 10 min and resuspended in 5 mL of PBS buffer. VgrGs, it is also possible that TEC proteins facilitate the binding Cell lysates were prepared by sonication, and cell debris was removed by of VgrG and effectors by presenting the binding partners in right centrifugation at 20,000 × g for 20 min. Dynabeads Protein G (Life Technol- conformation or maintaining protein stability. Structural analy- ogies) was incubated with 5 μg of monoclonal antibodies to 6His or V5 for 2 h ses of TEC, VgrG, and effector proteins are required to fully at 4 °C, and then mixed with 1 mL of cell lysates for 3 h at 4 °C. The magnetic understand not only the actions of TEC but also the mechanisms beads were washed three times with ice-cold PBST (phosphate saline buffer μ of T6SS effector delivery. with vol/vol 0.2% Tween 20) and then incubated with 30 L of SDS-loading Here, we report a class of diverse and TEC-dependent T6SS buffer, followed by incubation at 70 °C for 10 min to elute bound proteins. effectors. Future studies characterizing the functions of these Eluted samples were subject to Western blotting analysis as described above. effectors in different model systems will greatly increase our un- derstanding of the physiological role that T6SS plays in these Bioinformatic Analysis. Protein sequences were retrieved from the National Center for Biotechnology Information (NCBI) database and analyzed using species. Given the diverse ecological niches that these species HHpred (43) and Phyre2 (44, 45) for functional prediction. Representative occupy in the environment and the host, more novel functions of DUF4123 protein accession numbers and species were downloaded from the T6SS effectors are likely to be discovered. Because T6SS effectors Pfam protein database (39). Species carrying the DUF4123 domain, VipA are known to target essential cellular functions, including the cell (DFU770), and Hcp (DUF796) were downloaded from the Interpro database wall, membrane, and DNA/RNA of bacteria and the membrane and compared using the Gene List Venn Diagram program (genevenn. and cytoskeleton of eukaryotic cells (2, 4), the toxicity of effectors sourceforge.net/). Using the Pfam-generated species tree of DUF4123, we may provide an alternative therapeutic approach for treating selected representative species from each genus with fully annotated ge- bacterial infections or killing specific types of eukaryotic cells. nomes to characterize the DUF4123 immediate upstream and downstream proteins using the protein annotation in the NCBI database. Materials and Methods Bacterial Strains and Growth Conditions. Strains and plasmids used in this ACKNOWLEDGMENTS. We thank Drs. John Mekalanos, Thomas G. Bernhardt, study are listed in Table S1. Cultures were routinely grown aerobically at 37 °C and Nick Peters for providing bacterial strains. We thank Dr. Weilong Hao in LB (wt/vol 1% tryptone, 0.5% yeast extract, and 0.5% NaCl). Antibiotics for bioinformatics support. We thank Alex Le and other members of the T.G.D. laboratory for providing reagents and general support. This work was and chemicals were used at the following concentrations: ampicillin (100 μg/mL), μ μ μ supported by a start-up grant of the University of Calgary (to T.G.D.). T.G.D. streptomycin (100 g/mL), kanamycin (50 g/mL), tetracycline (10 g/mL), is supported by a Canada Research Chair award, a Canada Foundation for μ μ chloramphenicol (25 g/mL for E. coli,2.5 g/mL for SSU and V52), isopropyl Innovation-John R. Evans Leaders Fund grant, and an Alberta Innovation and β-D-1-thiogalactopyranoside (IPTG) (1 mM), and arabinose (wt/vol 0.1%). Mu- Advanced Education grant. M.W. is supported by a Cystic Fibrosis Canada tants of SSU and V52 were constructed using crossover PCR and homologous Postdoctoral Fellowship.

Liang et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 1. Fronzes R, Christie PJ, Waksman G (2009) The structural biology of type IV secretion 33. Zheng J, Ho B, Mekalanos JJ (2011) Genetic analysis of anti-amoebae and anti-bacterial systems. Nat Rev Microbiol 7(10):703–714. activities of the type VI secretion system in Vibrio cholerae. PLoS ONE 6(8):e23876. 2. Ho BT, Dong TG, Mekalanos JJ (2014) A view to a kill: The bacterial type VI secretion 34. Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system system. Cell Host Microbe 15(1):9–21. based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 95(10): 3. Hood RD, et al. (2010) A type VI secretion system of Pseudomonas aeruginosa targets 5752–5756. – a toxin to bacteria. Cell Host Microbe 7(1):25 37. 35. Ma AT, McAuley S, Pukatzki S, Mekalanos JJ (2009) Translocation of a Vibrio cholerae 4. Russell AB, Peterson SB, Mougous JD (2014) Type VI secretion system effectors: Poi- type VI secretion effector requires bacterial endocytosis by host cells. Cell Host Mi- – sons with a purpose. Nat Rev Microbiol 12(2):137 148. crobe 5(3):234–243. 5. Leiman PG, et al. (2009) Type VI secretion apparatus and phage tail-associated protein 36. Kitaoka M, Miyata ST, Brooks TM, Unterweger D, Pukatzki S (2011) VasH is a tran- complexes share a common evolutionary origin. Proc Natl Acad Sci USA 106(11): scriptional regulator of the type VI secretion system functional in endemic and 4154–4159. pandemic Vibrio cholerae. J Bacteriol 193(23):6471–6482. 6. Pukatzki S, et al. (2006) Identification of a conserved bacterial protein secretion sys- 37. Bernard CS, Brunet YR, Gavioli M, Lloubès R, Cascales E (2011) Regulation of type VI tem in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci secretion gene clusters by sigma54 and cognate enhancer binding proteins. JBacteriol USA 103(5):1528–1533. – 7. Mougous JD, et al. (2006) A virulence locus of Pseudomonas aeruginosa encodes a 193(9):2158 2167. protein secretion apparatus. Science 312(5779):1526–1530. 38. Dong TG, Mekalanos JJ (2012) Characterization of the RpoN regulon reveals differ- 8. Bingle LE, Bailey CM, Pallen MJ (2008) Type VI secretion: A beginner’s guide. Curr ential regulation of T6SS and new flagellar operons in Vibrio cholerae O37 strain V52. – Opin Microbiol 11(1):3–8. Nucleic Acids Res 40(16):7766 7775. 9. Fu Y, Waldor MK, Mekalanos JJ (2013) Tn-Seq analysis of Vibrio cholerae intestinal 39. Finn RD, et al. (2014) Pfam: The protein families database. Nucleic Acids Res 42(Database colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell issue, D1):D222–D230. Host Microbe 14(6):652–663. 40. Mougous JD, Gifford CA, Ramsdell TL, Mekalanos JJ (2007) Threonine phosphoryla- 10. Ma L-S, Hachani A, Lin J-S, Filloux A, Lai E-M (2014) Agrobacterium tumefaciens de- tion post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat ploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial Cell Biol 9(7):797–803. competition in planta. Cell Host Microbe 16(1):94–104. 41. Hachani A, Allsopp LP, Oduko Y, Filloux A (2014) The VgrG proteins are “à la carte” 11. Russell AB, et al. (2014) A type VI secretion-related pathway in Bacteroidetes mediates delivery systems for bacterial type VI effectors. J Biol Chem 289(25):17872–17884. interbacterial antagonism. Cell Host Microbe 16(2):227–236. 42. Suarez G, et al. (2008) Molecular characterization of a functional type VI secretion 12. Kudryashev M, et al. (2015) Structure of the type VI secretion system contractile system from a clinical isolate of Aeromonas hydrophila. Microb Pathog 44(4):344–361. sheath. Cell 160(5):952–962. 43. Söding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein ho- 13. Basler M, Pilhofer M, Henderson GP, Jensen GJ, Mekalanos JJ (2012) Type VI secretion mology detection and structure prediction. Nucleic Acids Res 33(Web Server issue): – requires a dynamic contractile phage tail-like structure. Nature 483:182 186. W244–W248. 14. Shneider MM, et al. (2013) PAAR-repeat proteins sharpen and diversify the type VI 44. Kelley LA, Sternberg MJE (2009) Protein structure prediction on the Web: A case study – secretion system spike. Nature 500(7462):350 353. using the Phyre server. Nat Protoc 4(3):363–371. 15. Basler M, Mekalanos JJ (2012) Type 6 secretion dynamics within and between bac- 45. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web terial cells. Science 337(6096):815. portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858. 16. Bönemann G, Pietrosiuk A, Diemand A, Zentgraf H, Mogk A (2009) Remodelling of 46. Meusch D, et al. (2014) Mechanism of Tc toxin action revealed in molecular detail. VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. Nature 508(7494):61–65. EMBO J 28(4):315–325. 47. Waterfield NR, Bowen DJ, Fetherston JD, Perry RD, ffrench-Constant RH (2001) The tc 17. Filloux A, Hachani A, Bleves S (2008) The bacterial type VI secretion machine: Yet genes of Photorhabdus: A growing family. Trends Microbiol 9(4):185–191. another player for protein transport across membranes. Microbiology 154(Pt 6): 48. Sha J, et al. (2013) Evaluation of the roles played by Hcp and VgrG type 6 secretion 1570–1583. system effectors in Aeromonas hydrophila SSU pathogenesis. Microbiology 159(Pt 6): 18. Dong TG, Ho BT, Yoder-Himes DR, Mekalanos JJ (2013) Identification of T6SS- – dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc Natl 1120 1135. – Acad Sci USA 110(7):2623–2628. 49. Cascales E, et al. (2007) Colicin biology. Microbiol Mol Biol Rev 71(1):158 229. 19. Brooks TM, Unterweger D, Bachmann V, Kostiuk B, Pukatzki S (2013) Lytic activity of 50. Palmer T, Berks BC (2012) The twin-arginine translocation (Tat) protein export – the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB. pathway. Nat Rev Microbiol 10(7):483 496. J Biol Chem 288(11):7618–7625. 51. Berks BC (1996) A common export pathway for proteins binding complex redox co- 20. Koskiniemi S, et al. (2013) Rhs proteins from diverse bacteria mediate intercellular factors? Mol Microbiol 22(3):393–404. competition. Proc Natl Acad Sci USA 110(17):7032–7037. 52. Silverman JM, et al. (2013) Haemolysin coregulated protein is an exported receptor 21. Pukatzki S, Ma AT, Revel AT, Sturtevant D, Mekalanos JJ (2007) Type VI secretion and chaperone of type VI secretion substrates. Mol Cell 51(5):584–593. system translocates a phage tail spike-like protein into target cells where it cross-links 53. King J, Wood WB (1969) Assembly of bacteriophage T4 tail fibers: The sequence of actin. Proc Natl Acad Sci USA 104(39):15508–15513. gene product interaction. J Mol Biol 39(3):583–601. 22. Suarez G, et al. (2010) A type VI secretion system effector protein, VgrG1, from 54. Bishop RJ, Wood WB (1976) Genetic analysis of T4 tail fiber assembly. I. A gene 37 Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin. mutation that allows bypass of gene 38 function. Virology 72(1):244–254. J Bacteriol 192(1):155–168. 55. Wood WB, Conley MP, Lyle HL, Dickson RC (1978) Attachment of tail fibers in bac- 23. Miyata ST, Unterweger D, Rudko SP, Pukatzki S (2013) Dual expression profile of type teriophage T4 assembly: Purification, properties, and site of action of the accessory VI secretion system immunity genes protects pandemic Vibrio cholerae. PLoS Pathog protein coded by gene 63. J Biol Chem 253(7):2437–2445. 9(12):e1003752. 56. Snopek TJ, Wood WB, Conley MP, Chen P, Cozzarelli NR (1977) Bacteriophage T4 RNA 24. Russell AB, et al. (2011) Type VI secretion delivers bacteriolytic effectors to target cells. ligase is gene 63 product, the protein that promotes tail fiber attachment to the – Nature 475(7356):343 347. baseplate. Proc Natl Acad Sci USA 74(8):3355–3359. 25. Dong TG, et al. (2015) Generation of reactive oxygen species by lethal attacks from 57. Vanderslice RW, Yegian CD (1974) The identification of late bacteriophage T4 pro- – competing microbes. Proc Natl Acad Sci USA 112(7):2181 2186. teins on sodium dodecyl sulfate polyacrylamide gels. Virology 60(1):265–275. 26. Dwyer DJ, et al. (2014) Antibiotics induce redox-related physiological alterations as 58. Page A-L, Parsot C (2002) Chaperones of the type III secretion pathway: Jacks of all part of their lethality. Proc Natl Acad Sci USA 111(20):E2100–E2109. trades. Mol Microbiol 46(1):1–11. 27. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ (2007) A common mech- 59. Ghosh P (2004) Process of protein transport by the type III secretion system. Microbiol anism of cellular death induced by bactericidal antibiotics. Cell 130(5):797–810. Mol Biol Rev 68(4):771–795. 28. Whitney JC, et al. (2014) Genetically distinct pathways guide effector export through 60. Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of the type VI secretion system. Mol Microbiol 92(3):529–542. insertion mutations: Osmoregulation of outer membrane proteins and virulence 29. Altindis E, Dong T, Catalano C, Mekalanos J (2015) Secretome analysis of Vibrio – cholerae type VI secretion system reveals a new effector-immunity pair. MBio 6(2): determinants in Vibrio cholerae requires toxR. J Bacteriol 170(6):2575 2583. e00075–e15. 61. Philippe N, Alcaraz J-P, Coursange E, Geiselmann J, Schneider D (2004) Improvement 30. English G, et al. (2012) New secreted toxins and immunity proteins encoded within of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid 51(3): the Type VI secretion system gene cluster of Serratia marcescens. Mol Microbiol 86(4): 246–255. 921–936. 62. Dong T, Yu R, Schellhorn H (2011) Antagonistic regulation of motility and transcriptome 31. Salomon D, et al. (2014) Marker for type VI secretion system effectors. Proc Natl Acad expression by RpoN and RpoS in Escherichia coli. Mol Microbiol 79(2):375–386. Sci USA 111(25):9271–9276. 63. Yang DC, et al. (2011) An ATP-binding cassette transporter-like complex governs cell- 32. Russell AB, et al. (2012) A widespread bacterial type VI secretion effector superfamily wall hydrolysis at the bacterial cytokinetic ring. Proc Natl Acad Sci USA 108(45): identified using a heuristic approach. Cell Host Microbe 11(5):538–549. E1052–E1060.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1505317112 Liang et al. Downloaded by guest on October 1, 2021