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• W2956985272 abstract "•Contractile injection systems are syringe-like structures from bacteria•A contractile injection system called MACs targets insect and mouse cell lines•MACs kill cell lines by delivering an effector called Pne1 with nuclease activity Many bacteria interact with target organisms using syringe-like structures called contractile injection systems (CISs). CISs structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. In many cases, CISs mediate trans-kingdom interactions between bacteria and eukaryotes by delivering effectors to target cells. However, the specific effectors and their modes of action are often unknown. Here, we establish an ex vivo model to study an extracellular CIS (eCIS) called metamorphosis-associated contractile structures (MACs) that target eukaryotic cells. MACs kill two eukaryotic cell lines, fall armyworm Sf9 cells and J774A.1 murine macrophage cells, by translocating an effector termed Pne1. Before the identification of Pne1, no CIS effector exhibiting nuclease activity against eukaryotic cells had been described. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a step toward developing CISs as novel delivery systems for eukaryotic hosts. Many bacteria interact with target organisms using syringe-like structures called contractile injection systems (CISs). CISs structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. In many cases, CISs mediate trans-kingdom interactions between bacteria and eukaryotes by delivering effectors to target cells. However, the specific effectors and their modes of action are often unknown. Here, we establish an ex vivo model to study an extracellular CIS (eCIS) called metamorphosis-associated contractile structures (MACs) that target eukaryotic cells. MACs kill two eukaryotic cell lines, fall armyworm Sf9 cells and J774A.1 murine macrophage cells, by translocating an effector termed Pne1. Before the identification of Pne1, no CIS effector exhibiting nuclease activity against eukaryotic cells had been described. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a step toward developing CISs as novel delivery systems for eukaryotic hosts. Bacteria interact with eukaryotic organisms with outcomes ranging from pathogenic to beneficial. One mechanism used by bacteria to interact with eukaryotes is through contractile injection systems (CISs) (Taylor et al., 2018Taylor N.M.I. van Raaij M.J. Leiman P.G. Contractile injection systems of bacteriophages and related systems.Mol. Microbiol. 2018; 108: 6-15Crossref PubMed Scopus (70) Google Scholar). CISs are evolutionarily related to the tails of bacteriophages (bacterial viruses) and are composed of an inner tube surrounded by a contractile sheath, capped with a tail spike and a baseplate complex. CISs can be classified into two types, type 6 secretion systems (T6SSs) and extracellular CISs (eCISs), also known as phage tail-like bacteriocins or tailocins. While T6SSs reside within the bacterial cytoplasm and are anchored to the inner membrane of Gram-negative bacteria (Böck et al., 2017Böck D. Medeiros J.M. Tsao H.-F. Penz T. Weiss G.L. Aistleitner K. Horn M. Pilhofer M. In situ architecture, function, and evolution of a contractile injection system.Science. 2017; 357: 713-717Crossref PubMed Scopus (85) Google Scholar, Ho et al., 2014Ho B.T. Dong T.G. Mekalanos J.J. A view to a kill: the bacterial type VI secretion system.Cell Host Microbe. 2014; 15: 9-21Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), eCISs are released extracellularly by bacterial cell lysis and bind their target cell surface (Hurst et al., 2004Hurst M.R.H. Glare T.R. Jackson T.A. Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica.J. Bacteriol. 2004; 186: 5116-5128Crossref PubMed Scopus (93) Google Scholar, Shikuma et al., 2014Shikuma N.J. Pilhofer M. Weiss G.L. Hadfield M.G. Jensen G.J. Newman D.K. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.Science. 2014; 343: 529-533Crossref PubMed Scopus (166) Google Scholar, Yang et al., 2006Yang G. Dowling A.J. Gerike U. ffrench-Constant R.H. Waterfield N.R. Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.J. Bacteriol. 2006; 188: 2254-2261Crossref PubMed Scopus (119) Google Scholar). It has been speculated that eCISs may be an evolutionary intermediate between bacteriophages and T6SSs (Büttner et al., 2016Büttner C.R. Wu Y. Maxwell K.L. Davidson A.R. Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems.Proc. Natl. Acad. Sci. USA. 2016; 113: 10174-10179Crossref PubMed Scopus (29) Google Scholar). In both eCISs and T6SSs, contraction of the sheath drives the inner tube and tail spike through the target cell membrane, and both often deliver effectors to host cells. For example, an eCIS called Photorhabdus virulence cassettes (PVCs) injects an effector causing deamidation and transglutamination of the cell cytoskeleton (Vlisidou et al., 2019Vlisidou I. Hapeshi A. Healey J.R.J. Smart K. Yang G. Waterfield N.R. Photorhabdus virulence cassettes: extracellular multi-protein needle complexes for delivery of small protein effectors into host cells.bioRxiv. 2019; https://doi.org/10.1101/549964Crossref Google Scholar, Yang et al., 2006Yang G. Dowling A.J. Gerike U. ffrench-Constant R.H. Waterfield N.R. Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.J. Bacteriol. 2006; 188: 2254-2261Crossref PubMed Scopus (119) Google Scholar). In T6SSs, a number of effectors are described that specifically target eukaryotic cells (Lien and Lai, 2017Lien Y.-W. Lai E.-M. Type VI secretion effectors: methodologies and biology.Front. Cell. Infect. Microbiol. 2017; 7: 254Crossref PubMed Scopus (60) Google Scholar). The modes of action of these T6SS effectors include actin cross-linking in macrophages (Pukatzki et al., 2007Pukatzki S. Ma A.T. Revel A.T. Sturtevant D. Mekalanos J.J. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.Proc. Natl. Acad. Sci. USA. 2007; 104: 15508-15513Crossref PubMed Scopus (521) Google Scholar), interaction with microtubules for invasion of epithelial cells (Sana et al., 2015Sana T.G. Baumann C. Merdes A. Soscia C. Rattei T. Hachani A. Jones C. Bennett K.L. Filloux A. Superti-Furga G. et al.Internalization of.MBio. 2015; 6: e00712Crossref PubMed Scopus (96) Google Scholar), and disruption of the actin cytoskeleton of HeLa cells (Suarez et al., 2010Suarez G. Sierra J.C. Erova T.E. Sha J. Horneman A.J. Chopra A.K. A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin.J. Bacteriol. 2010; 192: 155-168Crossref PubMed Scopus (155) Google Scholar). However, to our knowledge, and until the present work, no CIS effectors (T6SSs or eCISs) targeting eukaryotic cells that possess nuclease activity have been described. One group of evolutionarily related CIS have been shown to mediate interactions with diverse eukaryotic organisms, including amoebas, grass grubs, wax moths, and wasps (Böck et al., 2017Böck D. Medeiros J.M. Tsao H.-F. Penz T. Weiss G.L. Aistleitner K. Horn M. Pilhofer M. In situ architecture, function, and evolution of a contractile injection system.Science. 2017; 357: 713-717Crossref PubMed Scopus (85) Google Scholar, Hurst et al., 2007Hurst M.R.H. Beard S.S. Jackson T.A. Jones S.M. Isolation and characterization of the Serratia entomophila antifeeding prophage.FEMS Microbiol. Lett. 2007; 270: 42-48Crossref PubMed Scopus (53) Google Scholar, Penz et al., 2010Penz T. Horn M. Schmitz-Esser S. The genome of the amoeba symbiont “Candidatus Amoebophilus asiaticus” encodes an afp-like prophage possibly used for protein secretion.Virulence. 2010; 1: 541-545Crossref PubMed Scopus (21) Google Scholar, Penz et al., 2012Penz T. Schmitz-Esser S. Kelly S.E. Cass B.N. Müller A. Woyke T. Malfatti S.A. Hunter M.S. Horn M. Comparative genomics suggests an independent origin of cytoplasmic incompatibility in Cardinium hertigii.PLoS Genet. 2012; 8: e1003012Crossref PubMed Scopus (100) Google Scholar, Sarris et al., 2014Sarris P.F. Ladoukakis E.D. Panopoulos N.J. Scoulica E.V. A phage tail-derived element with wide distribution among both prokaryotic domains: a comparative genomic and phylogenetic study.Genome Biol. Evol. 2014; 6: 1739-1747Crossref PubMed Scopus (49) Google Scholar, Yang et al., 2006Yang G. Dowling A.J. Gerike U. ffrench-Constant R.H. Waterfield N.R. Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.J. Bacteriol. 2006; 188: 2254-2261Crossref PubMed Scopus (119) Google Scholar). We recently described a related eCIS mediating a beneficial relationship between the Gram-negative bacterium Pseudoalteromonas luteoviolacea and a marine tubeworm, Hydroides elegans, hereafter Hydroides (Huang et al., 2012Huang Y. Callahan S. Hadfield M.G. Recruitment in the sea: bacterial genes required for inducing larval settlement in a polychaete worm.Sci. Rep. 2012; 2: 228Crossref PubMed Scopus (57) Google Scholar, Shikuma et al., 2014Shikuma N.J. Pilhofer M. Weiss G.L. Hadfield M.G. Jensen G.J. Newman D.K. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.Science. 2014; 343: 529-533Crossref PubMed Scopus (166) Google Scholar, Shikuma et al., 2016Shikuma N.J. Antoshechkin I. Medeiros J.M. Pilhofer M. Newman D.K. Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 10097-10102Crossref PubMed Scopus (44) Google Scholar). We called this eCIS from P. luteoviolacea MACs (metamorphosis-associated contractile structures), because they stimulate the metamorphosis of Hydroides (Shikuma et al., 2014Shikuma N.J. Pilhofer M. Weiss G.L. Hadfield M.G. Jensen G.J. Newman D.K. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.Science. 2014; 343: 529-533Crossref PubMed Scopus (166) Google Scholar). MACs are the first CIS discovered to form arrays of phage tail-like structures composed of ∼100 tails and often measure ∼1 μm in diameter. While MACs provide another example of CIS-eukaryote interactions, the range of hosts targeted by eCISs like MACs as well as the identity and mode of action of effectors that mediate these interactions remain poorly understood. To study the interaction between MACs and eukaryotic cells, we establish an ex vivo CIS cell line interaction model with insect and mammalian cell types. Using these systems, we identify a new MAC effector with nuclease activity that is responsible for cytotoxicity in both cell types that we call Pne1 (Pseudoalteromonas nuclease effector 1). Our results indicate that MACs can interact with a range of host cells and that a specific effector mediates killing of eukaryotic cells. To study MACs from P. luteoviolacea and test their effect on eukaryotic cells, we focused on an insect cell line from the fall armyworm Spodoptera frugiperda (Sf9), the closest relative to Hydroides where established cell lines are commercially available. Upon co-incubation of purified MACs, we observed the lysis of Sf9 cells within 48 h (Figure 1A). As a control, we included cell-free purifications from a strain lacking the MAC baseplate gene macB, which is unable to produce intact phage tail-like structures and multi-tailed arrays (Shikuma et al., 2014Shikuma N.J. Pilhofer M. Weiss G.L. Hadfield M.G. Jensen G.J. Newman D.K. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.Science. 2014; 343: 529-533Crossref PubMed Scopus (166) Google Scholar). When Sf9 cells were co-incubated with purifications from a ΔmacB strain or extraction buffer alone, the cells remained viable (Figures 1B and 1C). We quantified the activity of MACs against Sf9 cells by staining dead cells using a colorimetric stain, trypan blue, or by a fluorescent dual stain, fluorescein diacetate (FDA) and propidium iodide (PI), which stains live and dead cells, respectively. Using both methods, we observed cell death when cells were exposed to wild-type (WT) MACs, while death was not observed with purifications from a ΔmacB strain or the extraction buffer (Figures 1D–1H). Filtering extract from WT cells through a 0.45-μm filter abolished the cell killing effect (Figure 1I), consistent with the observation that MACs form >0.45-μm arrays (Shikuma et al., 2014Shikuma N.J. Pilhofer M. Weiss G.L. Hadfield M.G. Jensen G.J. Newman D.K. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.Science. 2014; 343: 529-533Crossref PubMed Scopus (166) Google Scholar). Our results suggest that MACs are capable of targeting and killing insect cells. We previously showed that a locus containing six genes (JF50_12590-JF50_12615) within the P. luteoviolacea genome is required for MACs to stimulate the metamorphosis of the tubeworm Hydroides, yet a mutant lacking all six genes is still able to produce intact MAC structures (Shikuma et al., 2016Shikuma N.J. Antoshechkin I. Medeiros J.M. Pilhofer M. Newman D.K. Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 10097-10102Crossref PubMed Scopus (44) Google Scholar). To determine whether MACs require this same locus for insect cell line killing, we tested whether P. luteoviolacea mutants lacking each of the six genes were deficient in MAC-mediated insect cell killing. Among those six genes, only a ΔJF50_12610 mutant was unable to cause cell death upon co-incubation with insect cells (Figures 2A–2G). These results were quantified and confirmed using trypan blue or FDA-PI staining (Figures 2K and 2L). When JF50_12610 was introduced back into its native chromosomal locus, the killing effect of MACs was restored (Figure 2H). Intriguingly, when we tested MACs from the ΔJF50_12610 mutant against larvae from Hydroides, we found that this strain was capable of stimulating metamorphosis at levels comparable to that of WT MACs (Figure 2M), suggesting that functional MAC structures are still present. To determine whether the ΔJF50_12610 strain produces intact MAC structures, we employed electron cryo-tomography (ECT). Upon inspection, MACs from WT and ΔJF50_12610 were indistinguishable, forming intact phage tail-like structures in both extended and contracted conformations (Figures 2N and 2O). In order to confirm that JF50_12610 is part of the MAC complex, we utilized protein identification by mass spectrometry on purified MAC extracts from WT P. luteoviolacea. In two independent experiments, we detected JF50_12610, which indicated that the protein is associated with the MAC complex (Figure S1A). To determine whether the ΔJF50_12610 phenotype was due to the differential production of MACs, we quantified MACs tagged with super-folder GFP by fluorescent microscopy and found no difference in quantity between WT and ΔJF50_12610 strains grown under identical conditions (Figure S1B). Our results show that JF50_12610 is required for MACs to kill insect cell lines yet does not affect the ability of MACs to stimulate tubeworm metamorphosis or the production of functional MACs. The genes within the JF50_12590-JF50_12615 locus required for tubeworm metamorphosis, but not insect cell killing, are the subject of a separate work. To determine the function of JF50_12610, we searched the 496-amino-acid-long protein for conserved domains and homologous proteins. We found that JF50_12610 contains a DNA or RNA nonspecific nuclease domain (Pfam: PF13930). Analysis with the Phyre2 protein prediction program (Kelley et al., 2015Kelley L.A. Mezulis S. Yates C.M. Wass M.N. Sternberg M.J.E. The Phyre2 web portal for protein modeling, prediction and analysis.Nat. Protoc. 2015; 10: 845-858Crossref PubMed Scopus (6145) Google Scholar) showed that residues 258–348 of JF50_12610 bear 20% identity to the nuclease Spd1 from Streptococcus pyogenes (Korczynska et al., 2012Korczynska J.E. Turkenburg J.P. Taylor E.J. The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes.Nucleic Acids Res. 2012; 40: 928-938Crossref PubMed Scopus (18) Google Scholar), and residues 267–348 bear 30% identity to the nuclease Sda1 also from S. pyogenes (Moon et al., 2016Moon A.F. Krahn J.M. Lu X. Cuneo M.J. Pedersen L.C. Structural characterization of the virulence factor Sda1 nuclease from Streptococcus pyogenes.Nucleic Acids Res. 2016; 44: 3946-3957Crossref PubMed Scopus (13) Google Scholar) (Figure 3A). Through these partial alignments, we identified a conserved glutamic acid at residue 328 corresponding to Glu164 of Spd1 and Glu225 of Sda1 that coordinate water molecules hydrating the magnesium in the enzyme’s active site (Korczynska et al., 2012Korczynska J.E. Turkenburg J.P. Taylor E.J. The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes.Nucleic Acids Res. 2012; 40: 928-938Crossref PubMed Scopus (18) Google Scholar, Moon et al., 2016Moon A.F. Krahn J.M. Lu X. Cuneo M.J. Pedersen L.C. Structural characterization of the virulence factor Sda1 nuclease from Streptococcus pyogenes.Nucleic Acids Res. 2016; 44: 3946-3957Crossref PubMed Scopus (13) Google Scholar). Consistent with its predicted function as a toxic effector against eukaryotic cells, the JF50_12610 protein is predicted to contain a nuclear localization signal (NLStradamus program; Nguyen Ba et al., 2009Nguyen Ba A.N. Pogoutse A. Provart N. Moses A.M. NLStradamus: a simple hidden Markov model for nuclear localization signal prediction.BMC Bioinformatics. 2009; 10: 202Crossref PubMed Scopus (385) Google Scholar), which typically targets proteins to the nucleus of eukaryotic cells. Based on the predicted function of JF50_12610 and the results below, we named this effector Pne1. To determine whether Pne1 possesses nuclease activity, we cloned the WT pne1 gene and a pne1-Glu328Ala mutant into an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector system with N-terminal 6xHis tag and purified both proteins by nickel affinity chromatography (Figure 3B). When co-incubated with circular DNA, linear DNA or RNA, Pne1 and Pne1-Glu328Ala both exhibited nuclease activity (Figures 3C–3E), whereas a control protein (GFP), cloned and purified under the same conditions, did not exhibit nuclease activity. Our data suggest that Pne1 is an RNA/DNA endonuclease. Based on similarities between Pne1 and the magnesium-dependent homologous proteins Spd1 and Sda1, we tested the nuclease activity of Pne1 protein in the presence of the divalent cation chelator, EDTA. Interestingly, Pne1 and the Pne1-Glu328Ala proteins were still functional in the presence of EDTA (Figures 3C–3E). To test whether Pne1 requires its nuclear localization signal or the conserved Glu328 for killing insect cells, we created P. luteoviolacea mutants lacking the predicted nuclear localization signal (residues 19–52) pne1-ΔNLS or with the pne1-Glu328Ala point mutation in their native chromosomal loci. Upon exposure to insect cells, MACs from the pne1-ΔNLS strain partially abolished the killing effect and MACs from the pne1-Glu328Ala strain were unable to kill insect cells when compared to WT MACs (Figures 3F, 3H, 3I, and 3L). Our results show that Pne1 possesses nuclease activity in vitro and that its nuclear localization signal may be partially responsible for the killing effect. While the Glu328Ala is not required for nuclease activity in vitro, this residue is necessary for MACs to kill insect cells. We are currently investigating how the Glu328Ala residue contributes to insect cell killing. To determine whether MACs are capable of targeting a broader range of eukaryotic cells, we tested the ability of MACs to kill mammalian cells. We chose the commonly used murine macrophage cell line J774A.1, as these immune cells often encounter microbial pathogens and their effectors. Upon exposure of J774A.1 cells to WT MACs, we observed cell death within 24 h (Figure 4A). In contrast, MACs from a Δpne1 and ΔmacB, or buffer alone did not exhibit cell killing (Figures 4B–4D). Quantification of cytotoxicity by lactate dehydrogenase (LDH) release assays confirmed the observed killing upon exposure to WT MACs and lack of killing upon exposure to MACs from the Δpne1 (Figure 4E). Taken together, our results show that MACs are capable of targeting and killing mammalian cells and that the cell-killing phenotype is dependent on the presence of Pne1. In this work, we establish an ex vivo interaction model between an eCIS and two eukaryotic cell lines. With this system, we determined that the bacterial protein Pne1 is a novel eCIS effector and possesses RNA/DNA endonuclease activity. To our knowledge, our work is the first to identify a CIS with a broad eukaryotic host range and identify the first eukaryotic-targeting CIS effector with nuclease activity. Here, we show Pne1-dependent cell death of insect and mammalian cell lines, yet we have also previously observed that MACs stimulate metamorphosis in the tubeworm Hydroides (Shikuma et al., 2014Shikuma N.J. Pilhofer M. Weiss G.L. Hadfield M.G. Jensen G.J. Newman D.K. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.Science. 2014; 343: 529-533Crossref PubMed Scopus (166) Google Scholar). While it is unclear why MACs possess an effector that kills eukaryotic cells, some symbiotic bacteria have been shown to use CISs to modulate their host range. For example, the nitrogen-fixing plant symbiont Rhizobium leguminosarum limits its host range to plants in the clover family by secreting proteins through a T6SS, while mutation of the imp gene cluster (encoding components of the T6SS) allowed the bacterium to form functional root nodules on pea plants, normally outside of its host range (Bladergroen et al., 2003Bladergroen M.R. Badelt K. Spaink H.P. Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion.Mol. Plant Microbe Interact. 2003; 16: 53-64Crossref PubMed Scopus (171) Google Scholar). In the environment, Pseudoalteromonas species are found in association with many marine invertebrates (Holmström and Kjelleberg, 1999Holmström C. Kjelleberg S. Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents.FEMS Microbiol. Ecol. 1999; 30: 285-293Crossref PubMed Scopus (520) Google Scholar) and might utilize MACs and Pne1 to antagonize specific eukaryotes, like bacterivorous ciliates, while also stimulating the metamorphosis of other eukaryotes like Hydroides. Several CIS nuclease effectors have been identified targeting bacterial cells, including Tde1 from the soil bacterium Agrobacterium tumefaciens (Ma et al., 2014Ma L.S. Hachani A. Lin J.S. Filloux A. Lai E.M. Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta.Cell Host Microbe. 2014; 16: 94-104Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), RhsA from Dickeya dandantii (Koskiniemi et al., 2013Koskiniemi S. Lamoureux J.G. Nikolakakis K.C. t’Kint de Roodenbeke C. Kaplan M.D. Low D.A. Hayes C.S. Rhs proteins from diverse bacteria mediate intercellular competition.Proc. Natl. Acad. Sci. USA. 2013; 110: 7032-7037Crossref PubMed Scopus (256) Google Scholar), RhsP from Vibrio parahaemolyticus (Jiang et al., 2018Jiang N. Tang L. Xie R. Li Z. Burkinshaw B. Liang X. Sosa D. Aravind L. Dong T. Zhang D. Zheng J. Vibrio parahaemolyticus RhsP represents a widespread group of pro-effectors for type VI secretion systems.Nat. Commun. 2018; 9: 3899Crossref PubMed Scopus (6) Google Scholar), and Tse7 from Pseudomonas aeruginosa (Pissaridou et al., 2018Pissaridou P. Allsopp L.P. Wettstadt S. Howard S.A. Mavridou D.A.I. Filloux A. The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors.Proc. Natl. Acad. Sci. USA. 2018; 115: 12519-12524Crossref PubMed Scopus (59) Google Scholar). However, Pne1 is the first CIS nuclease effector to our knowledge that targets eukaryotic organisms. Interestingly, the eukaryotic nuclear localization signal at the N terminus of Pne1 had a partial effect on its ability to kill insect cells, further suggesting its evolved role in targeting eukaryotic hosts. Our results show that Glu328 is not required for Pne1 nuclease activity in vitro. However, this residue is necessary for MACs to kill insect cells. Two hypotheses that might explain these results are that (1) the Pne1-Glu328Ala mutant is not loaded into MACs and/or not translocated into insect cell lines properly or (2) Pne1-Glu328Ala is translocated, but the mutation renders it nontoxic. We will direct future experiments to determine the loading into MACs and/or translocation of Pne1 and Pne1-Glu328Ala to address these possibilities. While we show that Pne1 possesses DNA or RNA endonuclease activity, we currently do not know whether P. luteoviolacea protects itself from Pne1’s activity by means of an immunity protein. However, MACs differ from T6SS in that they are released by cell lysis extracellularly. Therefore, P. luteoviolacea may not require an immunity protein if, upon production of Pne1, they are programmed to lyse and release MACs. The host range of eukaryotic-targeting eCISs is currently poorly understood, and we are currently examining how MACs have different effects on other cell types and organisms. Intriguingly, work on related CISs show that many of them target eukaryotic organisms from diverse lineages, such as grass grubs, wax moths, wasps, and amoebas (Böck et al., 2017Böck D. Medeiros J.M. Tsao H.-F. Penz T. Weiss G.L. Aistleitner K. Horn M. Pilhofer M. In situ architecture, function, and evolution of a contractile injection system.Science. 2017; 357: 713-717Crossref PubMed Scopus (85) Google Scholar, Hurst et al., 2007Hurst M.R.H. Beard S.S. Jackson T.A. Jones S.M. Isolation and characterization of the Serratia entomophila antifeeding prophage.FEMS Microbiol. Lett. 2007; 270: 42-48Crossref PubMed Scopus (53) Google Scholar, Penz et al., 2010Penz T. Horn M. Schmitz-Esser S. The genome of the amoeba symbiont “Candidatus Amoebophilus asiaticus” encodes an afp-like prophage possibly used for protein secretion.Virulence. 2010; 1: 541-545Crossref PubMed Scopus (21) Google Scholar, Yang et al., 2006Yang G. Dowling A.J. Gerike U. ffrench-Constant R.H. Waterfield N.R. Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.J. Bacteriol. 2006; 188: 2254-2261Crossref PubMed Scopus (119) Google Scholar). While the ability of related eCISs, the anti-feeding prophage (Afp) and PVCs, to target eukaryotic cells has been attributed to tail fibers that resemble receptor-binding proteins from adenovirus (Hurst et al., 2007Hurst M.R.H. Beard S.S. Jackson T.A. Jones S.M. Isolation and characterization of the Serratia entomophila antifeeding prophage.FEMS Microbiol. Lett. 2007; 270: 42-48Crossref PubMed Scopus (53) Google Scholar, Yang et al., 2006Yang G. Dowling A.J. Gerike U. ffrench-Constant R.H. Waterfield N.R. Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.J. Bacteriol. 2006; 188: 2254-2261Crossref PubMed Scopus (119) Google Scholar), we detected no protein homology in MAC structures. We report here that MACs are capable of targeting multiple ex vivo cell lines from insect and mammalian lineages. By further studying how eCISs, like MACs, have evolved from bacteriophage origins to target eukaryotic cells, we can begin to determine the underlying mechanisms associated with these diverse interactions. In addition to expanding our basic understanding of CISs, our work opens the door for potentially using eCISs for biotechnology purposes. eCISs that target bacterial pathogens are already under development as narrow host-range antimicrobial agents (Scholl, 2017Scholl D. Phage tail-like bacteriocins.Annu. Rev. Virol. 2017; 4: 453-467Crossref PubMed Scopus (85) Google Scholar), for example against the gastrointestinal pathogen Clostridium difficile (Gebhart et al., 2015Gebhart D. Lok S. Clare S. Tomas M. Stares M. Scholl D. Donskey C.J. Lawley T.D. Govoni G.R. A modified R-type bacteriocin specifically targeting Clostridium difficile prevents colonization of mice without affecting gut microbiota diversity.MBio. 2015; 6 (e02368–e14)Crossref PubMed Scopus (81) Google Scholar). Because MACs are syringe-like structures that deliver proteinaceous cargo to eukaryotic cells, we are currently working to develop them as potential delivery systems for biotechnology applications. While the mechanism of any eCIS-eukaryotic cell attachment has yet to be determined, it is tantalizing to imagine using genetically modified CISs to deliver peptides of interest to specific eukaryotic cell types. The ex vivo system described in this work will significantly facilitate the realization of these efforts. Tabled 1REAGENT or RESOURCESOURCEIDENTIFIERBacterial and Virus StrainsSee Table S1.This PaperN/AChemicals, Peptides, and Recombinant ProteinsDulbecco’s Modified Eagle’s MediumGIBCO10566-016Fetal bovine serumThomas ScientificC838T67ESF 921 Insect Cell Culture MediumExpression systems96-001-01Luria-Bertani (LB) mediaThermo-FisherDF0446-17-3Instant O" @default.
• W2956985272 created "2019-07-23" @default.
• W2956985272 creator A5006903296 @default.
• W2956985272 creator A5034891144 @default.
• W2956985272 creator A5081412077 @default.
• W2956985272 creator A5084797728 @default.
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• W2956985272 date "2019-07-01" @default.
• W2956985272 modified "2023-09-26" @default.
• W2956985272 title "A Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector" @default.
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