Type VI secretion system MIX‐effectors carry both antibacterial and anti‐eukaryotic activities
2017; Springer Nature; Volume: 18; Issue: 11 Linguagem: Inglês
10.15252/embr.201744226
ISSN1469-3178
AutoresAnn Ray, Nika Schwartz, Marcela de Souza Santos, Junmei Zhang, Kim Orth, Dor Salomon,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoScientific Report14 September 2017Open Access Source DataTransparent process Type VI secretion system MIX-effectors carry both antibacterial and anti-eukaryotic activities Ann Ray Ann Ray Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Nika Schwartz Nika Schwartz Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Marcela de Souza Santos Marcela de Souza Santos Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Junmei Zhang Junmei Zhang Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Kim Orth Kim Orth orcid.org/0000-0002-0678-7620 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Dor Salomon Corresponding Author Dor Salomon [email protected] orcid.org/0000-0002-2009-9453 Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ann Ray Ann Ray Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Nika Schwartz Nika Schwartz Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Marcela de Souza Santos Marcela de Souza Santos Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Junmei Zhang Junmei Zhang Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Kim Orth Kim Orth orcid.org/0000-0002-0678-7620 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Dor Salomon Corresponding Author Dor Salomon [email protected] orcid.org/0000-0002-2009-9453 Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Author Information Ann Ray1,2, Nika Schwartz3, Marcela Souza Santos1, Junmei Zhang1,2, Kim Orth1,2,4 and Dor Salomon *,3 1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 2Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA 3Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 4Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA *Corresponding author. Tel: +972 3 6408583; E-mail: [email protected] EMBO Reports (2017)18:1978-1990https://doi.org/10.15252/embr.201744226 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti-eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine-like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX-effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti-eukaryotic effectors. One of these is a MIX-effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX-effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors. Synopsis Type VI secretion systems are protein delivery apparatuses that mediate predominately bactericidal activities. This study shows that a Vibrio T6SS utilizes a versatile repertoire of effectors to target both competing bacteria and neighbouring eukaryotic cells. The Vibrio proteolyticus T6SS1 mediates bacterial killing under warm marine-like conditions. The Vibrio T6SS1 delivers a suite of effectors with predicted bactericidal and virulence activities. A CNF1-containing Vibrio T6SS1 MIX-effector induces actin rearrangements and morphological changes upon infection of macrophages. Introduction A common strategy used by bacteria to manipulate neighbors is to secrete proteins, termed toxins or effectors, via specialized protein secretion systems 1. Whereas some secretion systems secrete toxins to the extracellular space, others can deliver them directly into neighboring cells 1. The type VI secretion system (T6SS) is a contact-dependent protein delivery apparatus found in many Gram-negative bacteria 23. This system is composed of a tail-tube made of stacked hexameric rings of a protein called Hcp 3, which are capped by a spike complex made of a VgrG trimer 4 and a PAAR repeat-containing protein 56. The tail-tube is decorated with effector proteins that mediate the activity of the T6SS 7. Upon activation, a sheath-like structure engulfing the tail-tube contracts and propels the tail-tube, with the effectors that decorate it, out of the bacterial cell and into an adjacent recipient, where effectors are deployed 68. T6SSs are unique as they can directly deliver effectors into either bacterial or eukaryotic cells 910 and thus play a role in interbacterial competition or virulence, respectively 7. T6SS effectors come in two flavors: as domains fused to tail-tube components, or as proteins that bind to these tail-tube components either directly 1112 or via adapter proteins 13141516. As many T6SSs described to date mediate antibacterial activities, most effectors that have been characterized are bactericidal and carry various activities such as lipases 1718, nucleases 19, peptidoglycan hydrolases 20, pore-forming 21, and NAD(P)+ glycohydrolases 22. To prevent self-intoxication, these bactericidal effectors are encoded adjacent to cognate immunity genes 2023. Several T6SSs were shown to target eukaryotic cells, yet compared to the number of bactericidal effectors, few anti-eukaryotic effectors have been described to date 24. These include toxin domains fused to "evolved VgrGs" 425, pore-forming VasX 2126, PldB phospholipase 27, TecA deamidase 28, EvpP 29, and effectors secreted by the Francisella T6SS 30. We recently uncovered a widespread, polymorphic class of T6SS effectors that share an N-terminal domain named MIX (Marker for type sIX effectors) and variable C-terminal toxin domains 31. MIX-effectors group into five clans named MIX I–V 31. Members of MIX V clan are horizontally shared between marine bacteria and are suggested to enhance their fitness 32. In a recent report, we determined the secretome of Vibrio proteolyticus (hereafter referred to as Vpr), a marine bacterium that has been previously isolated from diseased corals 3334. We identified a functional hemolysin that is cytotoxic toward mammalian cells 35. The Vpr secretome also included tail-tube components of T6SSs and MIX-effectors, indicating the presence of functional T6SSs. Here, we set out to characterize the T6SSs of Vpr and determine their effector repertoires and activities. We found that of the three T6SSs encoded by Vpr, T6SS1 mediated antibacterial activities. Using comparative proteomics analysis to identify the T6SS1 effector repertoire, combined with functional assays, we found that in addition to antibacterial MIX- and non-MIX-effectors, T6SS1 also secrets MIX-effectors with anti-eukaryotic activities. Thus, Vpr employs T6SS1 and a suite of MIX-effectors to manipulate both bacterial and eukaryotic neighbors. Results and Discussion Vpr encodes three T6SSs and seven MIX-effectors Genomic analysis of the Vpr strain ATCC 15338 (also called NBRC 13287) revealed three T6SSs encoded on three main gene clusters, each including the conserved core T6SS components and accessory genes 36 (Fig 1). The T6SS1 cluster (vpr01s_06_00050–vpr01s_6_00300) is homologous to the antibacterial Vibrio parahaemolyticus T6SS1 and Vibrio alginolyticus T6SS1, which we previously characterized 3132. It contains two putative transcription regulators (vpr01s_06_00080 and vpr01s_06_00230) 37 and a MIX-effector/immunity (E/I) pair (vpr01s_06_00050/vpr01s_06_00060) 31. However, it is missing the gene encoding a PAAR repeat-containing protein at the end of the cluster. The T6SS2 cluster (vpr01s_08_01740–vpr01s_08_01940) is similar to the T6SS3 cluster (vpr01s_01_04280–vpr01s_01_05020) in terms of gene content and organization, with two differences: (i) the genes encoding VgrG2-PAAR2 (vpr01s_08_01940–vpr01s_08_01920) are transcribed in the same direction as the rest of the cluster, whereas their counterparts in T6SS3 (vpr01s_01_05000–vpr01s_01_05020) are inverted; (ii) the genes between clpV and fha differ between the clusters (vpr01s_08_01820–vpr01s_08_01800 in T6SS2; vpr01s_01_04890–vpr01s_01_04880 in T6SS3) (Fig 1). Neither the T6SS2 nor the T6SS3 clusters appear to encode effectors. Figure 1. T6SS clusters and modules identified in silico in Vpr Genes are represented by arrows indicating direction of translation. Locus tags (vpr01s_xx_xxxxx) shown above. Encoded proteins and known domains shown below. Download figure Download PowerPoint The Vpr genome also contains "orphan" T6SS modules and effectors found outside the main T6SS clusters. An "orphan" hcp4 (vpr01s_11_00460) encodes a protein 99% identical to Hcp1. Five "orphan" modules encode PAAR repeat-containing proteins which are either found upstream of putative antibacterial E/I pairs with predicted nuclease or lysozyme-like activities (vpr01s_23_00260, vpr01s_15_00320, and vpr01s_04_02460) or are fused to a putative C-terminal effector domain (vpr01s_01_01270 and vpr01s_06_01360). There are also six "orphan" MIX-effectors, in addition to the MIX-effector found in the T6SS1 cluster. vpr01s_06_01360 is a MIX II clan member that is fused to an N-terminal PAAR domain mentioned above. vpr01s_28_00230 contains two MIX domains belonging to clans IV and V. vpr01s_06_01710 and vpr01s_25_00650 belong to the MIX V clan and carry putative antibacterial pore-forming (amino acid 502–584 similar to TcdA/TcdB pore-forming toxin domain with 46% probability, according to HHpred 38) and Pyocin_S nuclease (according to NCBI conserved domains) toxin domains, respectively. Interestingly, two "orphan" MIX-effectors belonging to the MIX V clan appear to be anti-eukaryotic rather than antibacterial. vpr01s_11_01570 contains a virulence CNF1 (cytotoxic necrotizing factor 1) domain, whereas vpr01s_11_01580 does not contain a known C-terminal domain but is not part of an E/I pair as it has no adjacent immunity gene. An additional putative antibacterial effector with a DUF2235 lipase domain 18 is encoded by vpr01s_10_00320 followed by three homologous putative immunity genes (vpr01s_10_00310–vpr01s_10_00290) (Fig 1). Interestingly, we found no "orphan" vgrG genes. Vpr kills competing bacteria under warm marine-like conditions To characterize the Vpr T6SSs, we first set out to identify conditions that activate them. As our analysis revealed several T6SS effectors with predicted bactericidal activities, we reasoned that at least one of the three Vpr T6SSs will mediate antibacterial toxicity, which we can monitor as an indicator of T6SS activity. Therefore, we performed bacterial competition assays and monitored the viability of Escherichia coli prey (which is commonly used as a Gram-negative prey in bacterial competition assays) when incubated together with Vpr under different temperature (23, 30, or 37°C) and salinity conditions (1% or 3% NaCl). We found that Vpr bactericidal activity against E. coli prey was most prominent at 30°C on media containing 3% NaCl, as under these conditions we observed the largest decrease in E. coli prey viability (Fig 2). Thus, Vpr kills competing bacteria preferentially under conditions that are found in marine environments during summer months. Figure 2. Vpr is bactericidal under warm marine-like conditions A, B. Viability counts of Escherichia coli prey before (0 h) and after (4 h) co-culture with wild-type Vpr or with no attacker (none) at indicated temperatures on media containing 1% (A) or 3% (B) NaCl. Download figure Download PowerPoint Vpr bactericidal activity is mediated by T6SS1 To determine whether the bactericidal activity of Vpr is mediated by either of the T6SSs, we generated strains deleted for each of the three vgrG genes that correspond to the three T6SS clusters. We then tested the ability of these mutants to kill E. coli prey. Inactivation of T6SS1 (ΔvgrG1) resulted in complete loss of bactericidal activity, whereas inactivation of T6SS2 (ΔvgrG2) or T6SS3 (ΔvgrG3) had no effect (Fig 3A). These results suggested that T6SS1 was responsible for the Vpr-mediated bactericidal activity seen in Fig 2. Indeed, the expression and secretion pattern of the secreted T6SS1 tail-tube spike component VgrG1 agreed with the level of Vpr bactericidal activity under the same conditions and peaked at 30°C in media containing 3% NaCl (Appendix Fig S1). Attempts to rescue the bactericidal activity of ΔvgrG1 by exogenous expression of VgrG1 from a plasmid were unsuccessful, possibly due to a polar effect of vgrG1 deletion on downstream T6SS1 components (Appendix Fig S2). Therefore, we generated another T6SS1 mutant in which we deleted the gene encoding the essential T6SS baseplate component TssG1 (ΔtssG1) 39. As with ΔvgrG1, the ΔtssG1 mutant lost bactericidal activity against E. coli (Fig 3B) as well as against V. parahaemolyticus, a marine bacterium that is found in the natural habitat of Vpr (Fig 3C). Exogenous expression of TssG1 from a plasmid rescued the bactericidal activity and the secretion of the spike component VgrG1 (Fig 3B–D). Notably, deletion of tssG1 had no adverse effects on Vpr growth (Appendix Fig S3). Thus, T6SS1 mediates the bactericidal activity of Vpr under warm marine-like conditions. Figure 3. T6SS1 mediates Vpr bactericidal activity A–C. Viability counts of Escherichia coli (A, B) and Vibrio parahaemolyticus POR1/Δhcp1 (C) prey before (0 h) and after (4 h) co-culture with indicated Vpr strains on media containing 3% NaCl at 30°C. Asterisks mark statistical significance between samples at 4-h timepoint by unpaired, two-tailed Student's t-test (*P < 0.05); n.s., no significant difference; WT, wild-type. D. Expression (Cells) and secretion (Media) of VgrG1 were detected by immunoblot using specific antibodies against VgrG1. Loading control (LC) is shown for total protein lysate. Data information: In (B–D), attackers contain either an empty expression vector (Empty) or vector for arabinose-inducible expression of TssG1 (pTssG1). Source data are available online for this figure. Source Data for Figure 3D [embr201744226-sup-0004-SDataFig3D.pdf] Download figure Download PowerPoint The Vpr T6SS1 secretome includes multiple tail-tube components and putative antibacterial and anti-eukaryotic effectors Next, we set out to determine the Vpr T6SS1 effectors that mediate the bactericidal activity. To this end, we used mass spectrometry to analyze and compare the secretomes of Vpr wild-type (T6SS1+) and ΔtssG1 (T6SS1−) grown under T6SS1-inducing conditions (i.e., 30°C in media containing 3% NaCl). We identified 27 proteins that were predominantly found in the secretome of the wild-type Vpr (ratio of wild-type/ΔtssG1 > 5) (Table 1 and Dataset EV1). Of these 27 proteins, 15 are predicted to be T6SS1 components and effectors (Fig 4), as they (i) are tail-tube components; (ii) contain MIX domains; (iii) are homologs of known T6SS effectors; (iv) contain predicted toxin domains; (v) do not contain a canonical secretion signal; (vi) are in an operon with T6SS tail-tube components. As expected, the tail-tube components Hcp1 and VgrG1, encoded within the T6SS1 gene cluster, were secreted in a T6SS1-dependent manner. Interestingly, the "orphan" Hcp4 and two "orphan" PAAR proteins (PAAR4 and PAAR6) were also identified as T6SS1 components 5. Table 1. T6SS1-dependent Vpr secretome Function/Role Uniprot Locus [vpr01s_] Description Domains Length [aa] Signal peptidea T6SS Tail-tube components U3BN98 06_00100 Hcp1 Hcp 172 No U3BB23 06_00110 VgrG1 VgrG 692 No U3BK42 11_00460 Hcp4 Hcp 172 No U3BHI2 23_00260 PAAR4 DUF4150 (PAAR) 503 No U2ZG25 04_02460 PAAR6 DUF4150 (PAAR) 942 No Antibacterial effectors U2ZJC6 10_00320 Lipase DUF2235 750 No U2ZYX6 04_02450 Nuclease HNH/ENDO VII 256 No U3A2L0 08_01700 Pore-forming TcdA/B pore-forming domain-like (TM) 356 No U3BK37 06_00050 MIX-effector MIX I 555 No U2ZN70 25_00650 MIX-effector nuclease MIX V, Pyocin_S 758 No U3BSB9 28_00230 MIX-effector MIX IV, LysM, MIX V (TM) 1,402 No Anti-eukaryotic effectors U3BEL6 11_01580 MIX-effector MIX V 591 No U3BNK6 11_01570 MIX-effector deamidase MIX V, CNF1 603 No U3A3W8 11_00400 Txp40 homolog 90 No Unknown U2ZZY6 06_00070 VP1390 homolog OmpA_C-like 1,539 No Non-T6SS Flagella U3B8Q8 03_01380 Flagellin Flagellin_N, Flagellin_C 288 No U2ZXS3 03_01390 Flagellar hook-associated protein 2 FliD_N, FliD_C 445 No U3B8S7 03_01530 Flagellar hook-associated protein 1 FlgK 457 No U3BHW0 03_01570 Flagellar basal body rod FlgG 261 No U2ZXU5 03_01590 Flagellar hook FlgE 398 No U3BHV5 03_01520 Flagellar hook-associated protein FlgL 299 No U2ZYS5 03_01510 Flagellin and related hook-associated protein FlgL 347 No Siderophore/heme transport U3BJL7 05_01130 Putative ferric siderophore receptor TonB-dependent receptor 663 Yes U3A4E3 17_00240 Ferrioxamine B receptor TonB-dependent receptor 710 Yes U2ZCG7 01_01640 Putative heme/hemoglobin receptor TonB-dependent receptor 711 Yes Other U3BAK7 05_01090 Hemolysin-type calcium-binding region homolog WD-like beta propeller repeat 463 Yes U3A3U4 11_00150 Protease Peptidase_M28 596 Yes TM, transmembrane. a As determined by SignalP 4.1. Figure 4. The Vpr T6SS1 secretome Schematic representation of putative Vpr T6SS1-secreted proteins identified by comparative proteomics (see Table 1). Horizontal black bars are relative to protein length. MIX domains and domains identified in NCBI CDD are illustrated in color; domain names are inside or below the rectangles. Transmembrane helices (TM) identified in UniProt are illustrated as white ovals with dashed perimeters. DUF4150 belong to PAAR-like superfamily; DUF2235 is a phospholipase. Download figure Download PowerPoint We also detected nine potential effectors in the T6SS1 secretome, including five MIX-effectors (Table 1 and Fig 4). Six are predicted antibacterial effectors with various toxin domains and downstream small open reading frames that likely encode cognate immunity proteins (Figs 1 and 4, and Appendix Fig S4). Vpr01s_10_00320 contains a DUF2235 lipase domain; Vpr01s_04_02450 is a HNH-family nuclease (99% probability according to HHpred 38 analysis) encoded downstream of PAAR6 (Fig 1); Vpr01s_08_01700 contains a C-terminal transmembrane region in a domain similar to pore-forming toxins (37% probability according to HHpred 38 analysis) (Table 1 and Fig 4) and is encoded close to the T6SS2 cluster (Appendix Fig S4). Three antibacterial effectors belong to the MIX-effector class: Vpr01s_06_00050 is encoded within the T6SS1 cluster and is homologous to the antibacterial MIX-effectors VP1388 and Va01565 from V. parahaemolyticus 31 and V. alginolyticus 32, respectively; Vpr01s_25_00650 belongs to the horizontally shared MIX V clan 32 and contains a Pyocin_S nuclease domain; Vpr01s_28_00230 contains both MIX IV and MIX V domains, as well as a C-terminal region homologous to that of the Vibrio cholerae pore-forming effector VasX 21 (Table 1 and Fig 4). Surprisingly, this bactericidal T6SS1 also secreted three effectors with putative anti-eukaryotic activities, which are not part of bicistronic units that encode potential immunity proteins (Figs 1 and 4). Vpr01s_11_01570 (named Vpr01570 hereafter) belongs to the horizontally shared MIX V clan 32 and contains a CNF1 deamidase domain that is predicted to target Rho GTPases 4041 (Appendix Fig S5). Vpr01s_11_01580 is encoded next to Vpr01570 and contains a MIX V domain (Fig 1). Its C-terminus is homologous to proteins encoded by bacteria of the genera Vibrio, Morganella, Pseudomonas, and Yersinia. Some of these homologs are annotated as cytotoxic and others contain Rhs repeats, which were suggested to be T6SS1 effectors 42. Vpr01s_11_00400 is a small protein homologous to the C-terminus of the insecticidal toxin Txp40 43 (Appendix Figs S4 and S6). Another T6SS1-secreted protein is Vpr01s_06_00070. It is encoded downstream of the MIX-E/I pair Vpr01s_06_00050/Vpr01s_06_00060 and is homologous to the V. parahaemolyticus VP1390, which we previously reported is secreted by the T6SS, yet its role remains unknown. Thus, the Vpr T6SS1 effector repertoire is diverse and includes multiple MIX-effectors with both antibacterial and anti-eukaryotic toxin domains. Notably, unlike the Vpr T6SS1, homologous systems in V. parahaemolyticus and V. alginolyticus secrete only one of each of the tail-tube components Hcp, VgrG, and PAAR 3132. Thus, it appears that the Vpr T6SS1 acquired additional "orphan" Hcp and PAAR proteins which possibly allow for diverse tail-tube combinations and enable secretion of a wide effector repertoire. The remaining 12 proteins predominantly found in the wild-type Vpr secretome are likely not direct substrates of T6SS1. These include flagellar components or proteins that contain a signal peptide and are associated with siderophore/heme transport, a protein containing a WD-like beta propeller repeat, and a protease (Table 1). While we do not yet know what mechanism leads to this apparent down-regulation of the flagellar machinery and other non-T6SS proteins, others have previously reported similar findings. For example, the T6SS in Citrobacter was reported to modulate flagellar gene expression and secretion 44, as did the T6SS in Ralstonia 45. Therefore, it is possible that a common cross talk mechanism exists between T6SS activity and motility, and perhaps also between the T6SS and siderophore or heme transport. The T6SS1 MIX-effector Vpr01570 is a functional CNF1 toxin Our comparative proteomics analysis revealed that T6SS1, which mediates bactericidal activities (Fig 3), also secretes effectors with predicted anti-eukaryotic activities (Table 1). This led us to hypothesize that the Vpr T6SS1 can use its diverse effector repertoire to target both bacteria and eukaryotic neighbors. To determine whether T6SS1 can also manipulate eukaryotic cells, we focused our efforts on the T6SS1 MIX-effector Vpr01570 that contains a CNF1 toxin domain and set out to monitor its functionality. CNF1 toxins are deamidases that target and activate Rho GTPases 4041. As Rho GTPases are master regulators of the actin cytoskeleton, CNF1 toxins induce actin cytoskeleton rearrangements inside eukaryotic cells, which are readily visualized by microscopy 41. We recently reported that Vpr induces cell death in human epithelial HeLa cells and in RAW 264.7 murine macrophages 35. This cytotoxicity was mediated by a secreted pore-forming hemolysin named VPRH 35. Strangely, however, unmasking this VPRH-mediated cytotoxicity by infecting HeLa cells with a Δvprh mutant did not reveal any observable actin cytoskeleton rearrangements 35. This result implied that the CNF1 MIX-effector is either not a functional toxin or is not delivered into HeLa cells during infection. We therefore tested whether Vpr01570 is a functional virulence toxin by exogenously expressing it in HeLa cells and in yeast. Expression of Vpr01570 fused to superfolder green fluorescent protein (sfGFP) induced dramatic actin cytoskeleton rearrangements in HeLa cells, including formation of actin stress fibers and ruffles which are hallmark phenotypes caused by CNF1 activity 40 (Fig EV1A). Vpr01570 was also toxic when expressed in yeast (Fig EV1B). These toxic effects were dependent on a functional CNF1 domain as they were not observed when a catalytic mutant (cysteine 450 to serine) (Appendix Fig S5) was expressed (Fig EV1A and B). Notably, expression of the Vpr01570 mutant form was detected in both HeLa cells and yeast, whereas the wild-type form was detected only in HeLa cells, presumably due to its deleterious effect in yeast (Fig EV1C and D). Moreover, we confirmed that deletion of vprh did not impair T6SS1 activity as a Δvprh mutant remained bactericidal in a bacterial competition assay (Appendix Fig S7). Thus, Vpr01570 is a functional CNF1 toxin, yet Vpr does not use T6SS1 or Vpr01570 to manipulate the actin cytoskeleton during infection of HeLa cells. Click here to expand this figure. Figure EV1. Vpr01570 is a functional anti-eukaryotic toxin A. Confocal micrograph of HeLa cells transfected with vectors expressing the indicated proteins. Cells were stained for F-actin and DNA using rhodamine-phalloidin (red) and Hoechst stain (blue), respectively. Scale bar = 30 μm; sfGFP, superfolder green fluorescent protein. Arrowheads mark actin ruffles. B. Growth of BY4741 yeast containing vectors for galactose-inducible expression of indicated proteins on repressing (glucose) and inducing (galactose) plates. Yeast were spotted in 10-fold serial dilutions. VopRΔ90/CA is a truncated and catalytically inactive form of the Vibrio parahaemolyticus type III effector VopR and serves as a non-toxic control. C, D. Immunoblot using anti-GFP (C) or anti-Myc (D) antibodies to verify expression of proteins used in (A) and (B), respectively. In (C), black arrow marks expected size of Vpr01570-sfGFP fusions and white arrow marks expected size of sfGFP. In (D), black arrow marks the expected size of Vpr01570-myc, and white arrow marks the expected size of VopRΔ90/CA-eGFP fusion. LC, loading control. Source data are available online for this figure. Download figure Download PowerPoint Vpr induces hemolysin-independent morphological changes in macrophages Previous reports suggested that the anti-eukaryotic activities of V.cholerae and Burkholderia pseudomallei T6SSs are activated upon internalization by phagocytic cells during infection 946. As we did not observe T6SS1-mediated morphological changes during infection of HeLa cells with the Δvprh mutant strain, we decided to test whether such effects will be detectable upon Vpr infection of phagocytic cells. To this end, we infected RAW 264.7 murine macrophages with Vpr. In agreement with our previous report 35, Vpr was cytotoxic to RAW 264.7 cells upon infection, and free nuclei from lysed cells were visible (Fig 5A). Remarkably, unmasking the VPRH-mediated cytotoxicity by infecting macrophages with a Δvprh mutant revealed dramatic morphological changes that were not detected upon infection of HeLa cells. RAW 264.7 cells infected with Δvprh became flat, spread out, developed actin ruffles on the top side, and showed a pronounced and smooth actin border starting at 4 h post-infection (Figs 5 and EV2). Notably, infection of macrophages with an avirulent derivative of the bacterium V. parahaemolyticus 47 did not induce similar phenotypes (Fig 5A), nor did incubation with lipopolysaccharide (LPS) (Fig EV2). These results suggested that the phenotypes observed during infection with the Vpr Δvprh mutant were induced by a Vpr determinant, and were not likely simply the result of macrophage activation by recognition of common pathogen-associated molecular patterns 48. Figure 5. The T6SS1 MIX-effector Vpr01570 induces actin rearrangements in macrophages Confocal micrograph of RAW 264.7 cells infected with indicated Vpr or avirulent Vibrio parahaeolyticus (V. para) strains for 4 h. Cells were stained for F-actin and DNA using rhodamine-phalloidin (yellow) and Hoechst stain (cyan), respectively. Top panels show bottom focal plane of the cells; lower panels show stacked z-axis slices of the same field of view. White arrows mark free nuclei of lysed cells.
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