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Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition
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Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition Nicole Kapitein and Axel Mogk Among specialized bacterial secretion systems, the most widespread is the type VI secretion system (T6SS). This transports effector molecules into target cells in a single, cellcontact dependent step. T6SSs are structurally related to the cell-puncturing device of tailed bacteriophages and predicted to function as contractile injection machineries that perforate eukaryotic and prokaryotic target membranes for effector delivery. Activities of T6SSs can play important roles in virulence by modifying the eukaryotic host cytoskeleton through actin crosslinking. They are also efficient weaponry in interbacterial warfare and provide a fitness advantage by hydrolyzing cell walls of opponent bacteria. The role of T6SSs in interbacterial competition might enable pathogens to outcompete commensal bacteria and facilitate host colonization.
of toxic effectors into target cells in a cell-contact dependent manner [1,6,7,8]. The export of hemolysin coregulated protein (Hcp) and valine–glycine repeat protein G (VgrG) proteins represents the unifying activity of all T6SSs. Apart from essential core components T6SSs also harbor accessory proteins, which allow for T6S regulation and host adaptation and might have led to evolvement of multiple, distinct T6SSs within several bacterial species.
Address Center for Molecular Biology of the University of Heidelberg and German Cancer Research Center, DKFZ-ZMBH Alliance, Universita¨t Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany
Contractile nanomachines: T6SSs puncture target membranes
Corresponding author: Mogk, Axel ([email protected])
Current Opinion in Microbiology 2013, 16:52–58 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Denise M Monack and Scott J Hultgren For a complete overview see the Issue and the Editorial Available online 2nd Jan 2013 1369-5274/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2012.11.009
Introduction The secretion of bacterial effector proteins into the extracellular space is a crucial step in pathogenesis and interbacterial virulence. In Gram-negative bacteria various specialized secretion systems mediate the transport of effector proteins in a single step across both the inner and outer membranes. In 2006, Mekalanos and coworkers identified the novel type VI secretion system (T6SS) [1,2], which has since been predicted in 25% of all sequenced Gram-negative bacteria, making T6SSs the most widespread specialized secretion system [3]. Some bacterial species even harbor multiple, distinct T6SSs. T6SSs are encoded by a gene cluster of 13 conserved core components that are all required for function [4,5]. Together these mediate the translocation Current Opinion in Microbiology 2013, 16:52–58
In this review, we will describe the current view of the mechanism and physiological functions of T6SSs. Because a universally accepted nomenclature for the T6SS components does not yet exist, we refer to the names used in the original studies.
Bioinformatic and structural analyses suggest that T6SSs are contractile injection systems and use a mechanism similar to tailed bacteriophages, which infect bacterial cells with high specificity and efficiency [9,10,11]. These bacteriophages employ a syringe-like macromolecular nanomachine to puncture the membrane of host cells. The infectious device consists of a baseplate, a contractile sheath that harbors an internal noncontractile tube and an associated tail spike complex [12]. Upon contact of the phage with the host cell surface conformational changes within the baseplate trigger contraction of the tail sheath, causing ejection of the internal tube and the tail spike complex and penetration of the host envelope [12] (Figure 1a). Several T6SS components exhibit sequence or structural similarity to the viral infection machinery. For example, Vibrio cholerae VCA0109 shares sequence homology with gp25, a component of the T4 bacteriophage baseplate [10]. The crystal structure of the T6SS exoprotein Hcp exhibits structural similarity to viral tail tube proteins [10,11]. Hcp forms hexameric rings with an outer diameter of 90 A˚ and a central channel with a width of 40 A˚ [2]. Hcp rings can form tubules in vitro if the individual stacks are stabilized by artificially introduced disulfide bonds [13]. The exoprotein VgrG forms a trimer that is highly similar to the viral tail spike complex, sharing a needle-like b-helix at the tip as the structural device to puncture target membranes [10,14]. The T6SS components VipA and VipB form tubular complexes up to 500 nm long, with an outer diameter of 300 A˚ and an inner channel diameter of 100 A˚ width [15]. These tubules are www.sciencedirect.com
Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition Kapitein and Mogk 53
reminiscent in shape and dimension to viral tail sheath proteins and have been suggested to engulf Hcp tubes and, in analogy to the viral infection mechanism, to eject Hcp and associated VgrG upon contraction [10,16]. VipA/VipB tubules have been visualized by electron cryotomography in intact V. cholerae cells [17]. The tubules are oriented near-perpendicular to the inner membrane and appear in an extended or contracted conformation. Extended structures appear to contain electron-dense material in the central channel, whereas contracted structures are hollow, providing indirect evidence for Hcp engulfment by VipA/VipB tubules [17]. VipA/VipB tubules are highly dynamic assemblies, showing cycles of polymerization, contraction, and disassembly in V. cholerae cells [17] (Figure 1b). Together these findings support the model that T6SSs function as contractile injection systems, sharing the mechanism of cell puncturing with tailed bacteriophages (Figure 1). One major difference between these contractile systems is that the injection machinery of bacteriophages is only used once and does not need to be recycled and reassembled for future infection rounds. In contrast, contracted VipA/ VipB tubules of T6SS are disassembled by the AAA+ protein ClpV under ATP consumption [15,18]. In V. cholerae ClpV exhibits a dynamic localization that is dependent on the presence of VipA/VipB tubules [18]. ClpV specifically binds to the contracted conformation of VipA/ VipB, restricting its disassembly activity to a post secretion step and allowing for multiple rounds of secretion and effector protein delivery (Figure 1b) [18]. ClpV dynamics and T6SS activity are reduced in Pseudomonas aeruginosa [18]. Here, T6SS activity is under additional posttranslational control, involving opposing activities of a kinase (PpkA) and phosphatase (PppA) pair that is controlling the phosphorylation status of the T6SS component Fha [19]. Kinase activity is suggested to be regulated by environmental cues, allowing to integrate external signals for activation of T6SS on demand [20]. Interestingly, an increase in T6SS activity was noticed in adjacent P. aeruginosa cells, suggesting that cell-to-cell signaling can modulate T6SS activity [18]. The syringe-like complex of T6SSs must be positioned and anchored to the bacterial cell envelope, in order to couple the energy from VipA/VipB contraction to transport across the inner and outer membranes. A membranespanning T6SS complex has been identified in enteroaggregative Escherichia coli (EAEC), consisting of TssL (DotU), TssM (IcmF), TssJ (SciN), and TagL (SciZ) [21]. TagL contains a peptidoglycan-binding domain, which is fused to TssL in other T6SSs, and anchors the T6SS assembly to the cell wall [21]. In EAEC TagL interacts with the inner membrane protein TssL, which forms a complex with TssM (IcmF), that also localizes to the inner membrane [21,22,23]. The periplasmic domain of TssM binds to the lipoprotein TssJ, which is anchored to the outer membrane [24]. This cell-envelope spanning www.sciencedirect.com
complex is suggested to accommodate the phage-like injection machinery. In support of such a model, interactions have been reported between VipA and Hcp and the periplasmic domain of TssM (IcmF) and TssL, respectively [4,25]. Notably, TssM (IcmF) is an essential ATPase of T6SSs and ATP hydrolysis is required for recruitment of Hcp to the membrane fraction, which potentially triggers Hcp polymerization [22,25].
Two routes of toxin delivery by T6SS The puncturing of target membranes by T6SS drives a path for toxin delivery. Two different classes of effectors have been characterized. The first class is represented by evolved VgrG proteins, which are present in various T6SSs, including the pathogenic genera Burkholderia, Pseudomonas, Yersinia and Vibrio [9]. Evolved VgrGs harbor an additional domain, which is C-terminally fused to the b-helix needle (Figure 2a) [9]. The fusion position ensures exposure of the extra domain upon perforation of target membranes. On the basis of sequence analysis distinct activities of evolved VgrGs have been predicted, including cell adhesion, chitosan degradation and actin filament binding and modification [9,26]. Translocation of the actin crosslinking domain of V. cholerae VgrG1 into target cells has been observed in cell culture systems and infant mice [9,27,28]. Aeromonas hydrophila VgrG1 exerts an actin ADP-ribosylation upon T6SS-dependent exposure in host cells [29]. Therefore, VgrG proteins have dual functions, and act as both integral structural components of the T6SS assembly and direct effectors. The second class of effectors is represented by classical toxins, which do not represent core components of T6SSs and are not required for Hcp and VgrG export [4,8,30,31]. Toxin encoding genes are often not directly associated with T6SS-encoding gene clusters. These effectors are thought to be delivered into target cells by passing through an Hcp channel. The inner diameter of the assumed Hcp conduit is wide enough (40 A˚) to allow for secretion of globular proteins with a size of up to approx. 50 kDa. However, the b-helix of associated VgrG trimers is too narrow to allow for effector passage, suggesting that VgrGs dissociate from Hcp tubes upon cell puncturing, which would free the block in the translocation channel. In agreement with this model, all classical T6SS effectors identified to date are smallsized proteins, except V. cholerae VasX [30,32].
Dual role of T6SS in pathogenicity and interbacterial competition The expression of T6SS encoding gene clusters in pathogenic bacteria is increased during infection [33–35] and is frequently regulated by environmental cues that mimic host conditions (for review see: [36]), which suggests a function in pathogenesis. The contributions of T6SSs to virulence development are diverse. In cell culture systems T6SSs have been reported to play crucial roles in Current Opinion in Microbiology 2013, 16:52–58
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Figure 1
(a) head
tail sheath
baseplate tail tube
tail spike
OM PG IM
(b)
VipA/B Contraction & Hcp/VgrG Ejection
OM TssJ
TssM (icmF)
PG TssL (DotU)
VgrG Hcp baseplate
IM VipA/B
VipA/B disintegration by CIpV
Reassembly 2
1 CIpV
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Tailed bacteriophages and T6SS share similar mechanisms of effector delivery by puncturing target membranes. Homologous and analogous viral and T6SS components are colored the same. (a) Binding of the bacteriophage to a bacterial host triggers contraction of tail sheath proteins. This leads to Current Opinion in Microbiology 2013, 16:52–58
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Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition Kapitein and Mogk 55
Figure 2
(a) Eukaryotic target cell:
(b) Prokaryotic target cell: OM PG IM Tse3
Tse1 Target cell
nucleus
ACD
Tse1
Tse3 Macrophage Tsi3
ACD
Tsi1 Pseudomonas aeruginosa
Vibrio cholerae
Tsi1
Tsi3 Tsi1
Tsi3
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Role of T6SS effector proteins in pathogenicity and interbacterial competition. (a) Upon puncturing of the eukaryotic host membrane, pathogenic V. cholerae evolved VgrG1 becomes exposed in the host cytosol and mediates actin crosslinking via its C-terminal ACD domain. VgrG1 may remain associated with the Hcp tube or becomes dissociated upon membrane perforation. (b) P. aeruginosa delivers the effector proteins Tse1 and Tse3 in a T6SS-dependent manner into the periplasm of bacterial target cells. The effectors are suggested to pass through an Hcp conduit. The amidase Tse1 and the muramidase Tse3 degrade the cell wall of target cells, causing cell lysis. Specific immunity proteins (Tsi1/Tsi3) present in the periplasm protect P. aeruginosa from self-killing. OM: outer membrane; IM: inner membrane; PG: peptidoglycan layer.
cell adhesion and invasion [37–40] and intracellular growth [41–43]. In Burkholderia sp. T6SSs play essential roles in pathogenicity [42,44]. Significant contributions to virulence have been reported in other cases including the fish pathogen Edwardsiella tarda and the plant pathogen Agrobacterium tumefaciens [4,45,46]. Immunization of animals with recombinant Hcp proteins can protect from subsequent infection by the pathogens [42,45]. In most cases the physiological targets of T6SSs have not been identified, except for V. cholerae. Here, the actin-crosslinking activity of VgrG1 is essential to cause inflammatory diarrhea in infant mice and rabbits [28,47] (Figure 2a). In other pathogens, T6SSs have minor or no contributions to pathogenicity [48,49]. In some cases, inactivation of a T6SS even increases virulence, suggesting that T6SSs might limit acute pathogenic activities in these bacteria [35,50]. For some bacterial species even an inverse correlation between pathogenicity and T6SS abundance was noticed [51]. Many bacteria harboring a T6SS are not pathogens, indicating additional physiological functions of T6SSs unassociated with virulence. T6SSs have now
been demonstrated to provide an effective weaponry against Gram-negative bacteria and unicellular eukaryotes. They were originally identified by a genetic screen in V. cholerae to isolate factors required to protect the bacteria from predation by the unicellular eukaryote Dictyostelium discoideum [1]. An antibacterial activity for T6SSs was first reported for P. aeruginosa by Mougous and coworkers and was also subsequently demonstrated for T6SSs of V. cholerae, Burkholderia thailandensis and Serratia marcescens [7,8,52,53]. The T6SS of Pseudomonas syringae allows it to survive in presence of Saccharomyces cerevisiae by suppressing growth of yeast cells in competition experiments [54]. The dual role of T6SSs in pathogenicity and interbacterial competition is best exemplified by the specialized T6SSs of B. thailandensis, which encodes for five T6SSs. In this organism one T6SS (T6SS-1) is required for growth in the presence of competing bacteria, whereas another T6SS (T6SS-5) is essential for virulence in a mouse infection model [52]. In P. aeruginosa, the antibacterial activity is provided by the peptidoglycan degrading effectors Tse1 and Tse3, which are targeted to the periplasm of prey cells in an
( Figure 1 Legend Continued ) the ejection of the tail tube and the associated tail spike complex and results in puncturing of the outer membrane of the bacterial host. This ultimately results in the delivery of the viral DNA stored in the head. (b) Hcp tubes and an associated VgrG trimer exhibit structural similarity to the viral cell puncturing device. Hcp tubes are suggested to be engulfed by extended VipA/VipB tubules, which function in analogy to viral tail sheath proteins. The complex is anchored to the cell envelope via a membrane-associated complex, composed of TssJ, TssM (IcmF) and TssL (DotU), and a baseplate-like structure. Contraction of VipA/VipB causes ejection of Hcp and VgrG. Ejected Hcp and VgrG might dismantle from the cell surface. Contracted VipA/VipB tubules are subsequently removed by the ATPase ClpV, allowing for a new round of Hcp, VgrG, and VipA/VipB assembly and thereby a new round of secretion. OM: outer membrane; IM: inner membrane; PG: peptidoglycan layer. www.sciencedirect.com
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H1-T6SS-dependent manner (Figure 2b) [6]. Tse1 and Tse3 exhibit different enzymatic activities, which may allow P. aeruginosa to kill a broader range of bacteria. The effector proteins are not linked to the T6SS-encoding gene cluster, but are coorganized with specific immunity proteins (e.g. Tse1), which protect P. aeruginosa cells from self killing by endogenous or intercellularly transferred toxins (Figure 2b) [6,8]. Using a heuristic approach 51 Tse1/Tsi1 toxin–antitoxin pairs were predicted in a broad range of proteobacterial species, all of them encoding for T6SSs [32]. A T6SS-dependent secretion of a predicted toxin family member and the presence of a periplasmic immunity protein for self-protection have been recently demonstrated for S. marcescens [55]. The predicted Tse1/ Tsi1 pairs were enriched in bacteria that face high densities of competing bacteria, for example, in soil or inside the gastrointestinal tract [32]. This suggests that T6SSs can increase bacterial fitness, allowing species that contain T6SSs to outcompete other bacteria. The antibacterial potential of T6SSs is also reflected by fusions of a peptidoglycan-binding domain or a LysM domain, showing structural homology to lysozyme, to V. cholerae or Pantoea agglomerans VgrGs [9,26]. Furthermore, S-type pyocins are fused to either S. enterica VgrG or uropathogenic E. coli Hcp, suggesting that the corresponding T6SSs deliver the bacteriocins to target cells upon membrane puncturing [56]. The advantageous effect of T6SSs in interbacterial competition may also have important supportive roles in polymicrobial infections. The human body harbors a complex and competitive microbiota and pathogens must displace commensal bacteria, which form a protective barrier preventing invader colonization. T6SSs can enable pathogens to kill off resident commensal bacteria, facilitating colonization of the host. This indirect role of T6SSs in virulence might have important impacts on virulence development and disease outcome [51]. T6SS activities are not only restricted to virulence toward eukaryotic and prokaryotic hosts but can also be involved in controlling host specificity [57] or biofilm formation [24,39]. T6SS components enable Proteus mirabilis cells to distinguish self from non-self [58]. The type VI secretion is a versatile toolbox, which enables Gram-negative bacteria to communicate with other cells in many contexts for diverse outputs.
Future directions It is now established that T6SSs form contractile machineries for membrane perforation and toxin delivery. Still, we are far from understanding the precise mechanism of T6S as many essential components remain poorly characterized. It is unclear whether classical effector are indeed passing an Hcp conduit and if so, how their transport is energized. Initial data indicate that T6SS activity can be regulated post-translationally allowing for the integration of environmental cues. The nature of these signals and Current Opinion in Microbiology 2013, 16:52–58
the signal transduction cascades are largely unknown. Is T6SS activation triggered by cell-to-cell contact? Does such regulation involve control of T6SS assembly or contraction of the VipA/VipB sheath structure? The dual role of T6SSs in pathogenicity and antibacterial virulence makes them an attractive target for muchneeded novel antibacterial therapeutics. Furthermore, surface-exposure of various T6SS components qualifies them as well-suited candidates for vaccination. The potential role of antibacterial T6SS activity in disease outcome is, however, still in need of documentation. Additional cellular targets of T6SSs in eukaryotic hosts remain to be identified. On the basis of bioinformatic analysis, potential functions of evolved VgrGs and classical effectors have been predicted, but remain to be confirmed. The rapid progress that has been achieved since the discovery of T6SSs holds great promise that these gaps can be filled soon.
Acknowledgements We thank Damon Huber, Gabriele Bo¨nemann and Lys Guilbride for editing of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A.M. (MO970/3). N.K. was supported by the Hartmut Hoffman-Berling International Graduate School of Molecular and Cellular Biology (HBIGS).
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51. Schwarz S, Hood RD, Mougous JD: What is type VI secretion doing in all those bugs? Trends Microbiol 2010, 18:531-537. 52. Schwarz S, West TE, Boyer F, Chiang WC, Carl MA, Hood RD, Rohmer L, Tolker-Nielsen T, Skerrett SJ, Mougous JD: Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog 2010, 6. The paper shows specific roles of distinct T6SSs present in one species in pathogenicity and interbacterial competition. 53. Murdoch SL, Trunk K, English G, Fritsch MJ, Pourkarimi E, Coulthurst SJ: The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors. J Bacteriol 2011, 193:6057-6069. 54. Haapalainen M, Mosorin H, Dorati F, Wu RF, Roine E, Taira S, Nissinen R, Mattinen L, Jackson R, Pirhonen M et al.: Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for competitive fitness against bacteria and yeasts. J Bacteriol 2012, 194:4810-4822. 55. English G, Trunk K, Rao VA, Srikannathasan V, Hunter WN, Coulthurst SJ: New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens. Mol Microbiol 2012 http://dx.doi.org/ 10.1111/mmi.12028. 56. Blondel CJ, Jimenez JC, Contreras I, Santiviago CA: Comparative genomic analysis uncovers 3 novel loci encoding type six secretion systems differentially distributed in Salmonella serotypes. BMC Genomics 2009, 10:354.
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Current Opinion in Microbiology 2013, 16:52–58
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Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition Nicole Kapitein and Axel Mogk Among specialized bacterial secretion systems, the most widespread is the type VI secretion system (T6SS). This transports effector molecules into target cells in a single, cellcontact dependent step. T6SSs are structurally related to the cell-puncturing device of tailed bacteriophages and predicted to function as contractile injection machineries that perforate eukaryotic and prokaryotic target membranes for effector delivery. Activities of T6SSs can play important roles in virulence by modifying the eukaryotic host cytoskeleton through actin crosslinking. They are also efficient weaponry in interbacterial warfare and provide a fitness advantage by hydrolyzing cell walls of opponent bacteria. The role of T6SSs in interbacterial competition might enable pathogens to outcompete commensal bacteria and facilitate host colonization.
of toxic effectors into target cells in a cell-contact dependent manner [1,6,7,8]. The export of hemolysin coregulated protein (Hcp) and valine–glycine repeat protein G (VgrG) proteins represents the unifying activity of all T6SSs. Apart from essential core components T6SSs also harbor accessory proteins, which allow for T6S regulation and host adaptation and might have led to evolvement of multiple, distinct T6SSs within several bacterial species.
Address Center for Molecular Biology of the University of Heidelberg and German Cancer Research Center, DKFZ-ZMBH Alliance, Universita¨t Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany
Contractile nanomachines: T6SSs puncture target membranes
Corresponding author: Mogk, Axel ([email protected])
Current Opinion in Microbiology 2013, 16:52–58 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Denise M Monack and Scott J Hultgren For a complete overview see the Issue and the Editorial Available online 2nd Jan 2013 1369-5274/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2012.11.009
Introduction The secretion of bacterial effector proteins into the extracellular space is a crucial step in pathogenesis and interbacterial virulence. In Gram-negative bacteria various specialized secretion systems mediate the transport of effector proteins in a single step across both the inner and outer membranes. In 2006, Mekalanos and coworkers identified the novel type VI secretion system (T6SS) [1,2], which has since been predicted in 25% of all sequenced Gram-negative bacteria, making T6SSs the most widespread specialized secretion system [3]. Some bacterial species even harbor multiple, distinct T6SSs. T6SSs are encoded by a gene cluster of 13 conserved core components that are all required for function [4,5]. Together these mediate the translocation Current Opinion in Microbiology 2013, 16:52–58
In this review, we will describe the current view of the mechanism and physiological functions of T6SSs. Because a universally accepted nomenclature for the T6SS components does not yet exist, we refer to the names used in the original studies.
Bioinformatic and structural analyses suggest that T6SSs are contractile injection systems and use a mechanism similar to tailed bacteriophages, which infect bacterial cells with high specificity and efficiency [9,10,11]. These bacteriophages employ a syringe-like macromolecular nanomachine to puncture the membrane of host cells. The infectious device consists of a baseplate, a contractile sheath that harbors an internal noncontractile tube and an associated tail spike complex [12]. Upon contact of the phage with the host cell surface conformational changes within the baseplate trigger contraction of the tail sheath, causing ejection of the internal tube and the tail spike complex and penetration of the host envelope [12] (Figure 1a). Several T6SS components exhibit sequence or structural similarity to the viral infection machinery. For example, Vibrio cholerae VCA0109 shares sequence homology with gp25, a component of the T4 bacteriophage baseplate [10]. The crystal structure of the T6SS exoprotein Hcp exhibits structural similarity to viral tail tube proteins [10,11]. Hcp forms hexameric rings with an outer diameter of 90 A˚ and a central channel with a width of 40 A˚ [2]. Hcp rings can form tubules in vitro if the individual stacks are stabilized by artificially introduced disulfide bonds [13]. The exoprotein VgrG forms a trimer that is highly similar to the viral tail spike complex, sharing a needle-like b-helix at the tip as the structural device to puncture target membranes [10,14]. The T6SS components VipA and VipB form tubular complexes up to 500 nm long, with an outer diameter of 300 A˚ and an inner channel diameter of 100 A˚ width [15]. These tubules are www.sciencedirect.com
Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition Kapitein and Mogk 53
reminiscent in shape and dimension to viral tail sheath proteins and have been suggested to engulf Hcp tubes and, in analogy to the viral infection mechanism, to eject Hcp and associated VgrG upon contraction [10,16]. VipA/VipB tubules have been visualized by electron cryotomography in intact V. cholerae cells [17]. The tubules are oriented near-perpendicular to the inner membrane and appear in an extended or contracted conformation. Extended structures appear to contain electron-dense material in the central channel, whereas contracted structures are hollow, providing indirect evidence for Hcp engulfment by VipA/VipB tubules [17]. VipA/VipB tubules are highly dynamic assemblies, showing cycles of polymerization, contraction, and disassembly in V. cholerae cells [17] (Figure 1b). Together these findings support the model that T6SSs function as contractile injection systems, sharing the mechanism of cell puncturing with tailed bacteriophages (Figure 1). One major difference between these contractile systems is that the injection machinery of bacteriophages is only used once and does not need to be recycled and reassembled for future infection rounds. In contrast, contracted VipA/ VipB tubules of T6SS are disassembled by the AAA+ protein ClpV under ATP consumption [15,18]. In V. cholerae ClpV exhibits a dynamic localization that is dependent on the presence of VipA/VipB tubules [18]. ClpV specifically binds to the contracted conformation of VipA/ VipB, restricting its disassembly activity to a post secretion step and allowing for multiple rounds of secretion and effector protein delivery (Figure 1b) [18]. ClpV dynamics and T6SS activity are reduced in Pseudomonas aeruginosa [18]. Here, T6SS activity is under additional posttranslational control, involving opposing activities of a kinase (PpkA) and phosphatase (PppA) pair that is controlling the phosphorylation status of the T6SS component Fha [19]. Kinase activity is suggested to be regulated by environmental cues, allowing to integrate external signals for activation of T6SS on demand [20]. Interestingly, an increase in T6SS activity was noticed in adjacent P. aeruginosa cells, suggesting that cell-to-cell signaling can modulate T6SS activity [18]. The syringe-like complex of T6SSs must be positioned and anchored to the bacterial cell envelope, in order to couple the energy from VipA/VipB contraction to transport across the inner and outer membranes. A membranespanning T6SS complex has been identified in enteroaggregative Escherichia coli (EAEC), consisting of TssL (DotU), TssM (IcmF), TssJ (SciN), and TagL (SciZ) [21]. TagL contains a peptidoglycan-binding domain, which is fused to TssL in other T6SSs, and anchors the T6SS assembly to the cell wall [21]. In EAEC TagL interacts with the inner membrane protein TssL, which forms a complex with TssM (IcmF), that also localizes to the inner membrane [21,22,23]. The periplasmic domain of TssM binds to the lipoprotein TssJ, which is anchored to the outer membrane [24]. This cell-envelope spanning www.sciencedirect.com
complex is suggested to accommodate the phage-like injection machinery. In support of such a model, interactions have been reported between VipA and Hcp and the periplasmic domain of TssM (IcmF) and TssL, respectively [4,25]. Notably, TssM (IcmF) is an essential ATPase of T6SSs and ATP hydrolysis is required for recruitment of Hcp to the membrane fraction, which potentially triggers Hcp polymerization [22,25].
Two routes of toxin delivery by T6SS The puncturing of target membranes by T6SS drives a path for toxin delivery. Two different classes of effectors have been characterized. The first class is represented by evolved VgrG proteins, which are present in various T6SSs, including the pathogenic genera Burkholderia, Pseudomonas, Yersinia and Vibrio [9]. Evolved VgrGs harbor an additional domain, which is C-terminally fused to the b-helix needle (Figure 2a) [9]. The fusion position ensures exposure of the extra domain upon perforation of target membranes. On the basis of sequence analysis distinct activities of evolved VgrGs have been predicted, including cell adhesion, chitosan degradation and actin filament binding and modification [9,26]. Translocation of the actin crosslinking domain of V. cholerae VgrG1 into target cells has been observed in cell culture systems and infant mice [9,27,28]. Aeromonas hydrophila VgrG1 exerts an actin ADP-ribosylation upon T6SS-dependent exposure in host cells [29]. Therefore, VgrG proteins have dual functions, and act as both integral structural components of the T6SS assembly and direct effectors. The second class of effectors is represented by classical toxins, which do not represent core components of T6SSs and are not required for Hcp and VgrG export [4,8,30,31]. Toxin encoding genes are often not directly associated with T6SS-encoding gene clusters. These effectors are thought to be delivered into target cells by passing through an Hcp channel. The inner diameter of the assumed Hcp conduit is wide enough (40 A˚) to allow for secretion of globular proteins with a size of up to approx. 50 kDa. However, the b-helix of associated VgrG trimers is too narrow to allow for effector passage, suggesting that VgrGs dissociate from Hcp tubes upon cell puncturing, which would free the block in the translocation channel. In agreement with this model, all classical T6SS effectors identified to date are smallsized proteins, except V. cholerae VasX [30,32].
Dual role of T6SS in pathogenicity and interbacterial competition The expression of T6SS encoding gene clusters in pathogenic bacteria is increased during infection [33–35] and is frequently regulated by environmental cues that mimic host conditions (for review see: [36]), which suggests a function in pathogenesis. The contributions of T6SSs to virulence development are diverse. In cell culture systems T6SSs have been reported to play crucial roles in Current Opinion in Microbiology 2013, 16:52–58
54 Host–microbe interactions: bacteria
Figure 1
(a) head
tail sheath
baseplate tail tube
tail spike
OM PG IM
(b)
VipA/B Contraction & Hcp/VgrG Ejection
OM TssJ
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PG TssL (DotU)
VgrG Hcp baseplate
IM VipA/B
VipA/B disintegration by CIpV
Reassembly 2
1 CIpV
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Tailed bacteriophages and T6SS share similar mechanisms of effector delivery by puncturing target membranes. Homologous and analogous viral and T6SS components are colored the same. (a) Binding of the bacteriophage to a bacterial host triggers contraction of tail sheath proteins. This leads to Current Opinion in Microbiology 2013, 16:52–58
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Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition Kapitein and Mogk 55
Figure 2
(a) Eukaryotic target cell:
(b) Prokaryotic target cell: OM PG IM Tse3
Tse1 Target cell
nucleus
ACD
Tse1
Tse3 Macrophage Tsi3
ACD
Tsi1 Pseudomonas aeruginosa
Vibrio cholerae
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Tsi3 Tsi1
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Role of T6SS effector proteins in pathogenicity and interbacterial competition. (a) Upon puncturing of the eukaryotic host membrane, pathogenic V. cholerae evolved VgrG1 becomes exposed in the host cytosol and mediates actin crosslinking via its C-terminal ACD domain. VgrG1 may remain associated with the Hcp tube or becomes dissociated upon membrane perforation. (b) P. aeruginosa delivers the effector proteins Tse1 and Tse3 in a T6SS-dependent manner into the periplasm of bacterial target cells. The effectors are suggested to pass through an Hcp conduit. The amidase Tse1 and the muramidase Tse3 degrade the cell wall of target cells, causing cell lysis. Specific immunity proteins (Tsi1/Tsi3) present in the periplasm protect P. aeruginosa from self-killing. OM: outer membrane; IM: inner membrane; PG: peptidoglycan layer.
cell adhesion and invasion [37–40] and intracellular growth [41–43]. In Burkholderia sp. T6SSs play essential roles in pathogenicity [42,44]. Significant contributions to virulence have been reported in other cases including the fish pathogen Edwardsiella tarda and the plant pathogen Agrobacterium tumefaciens [4,45,46]. Immunization of animals with recombinant Hcp proteins can protect from subsequent infection by the pathogens [42,45]. In most cases the physiological targets of T6SSs have not been identified, except for V. cholerae. Here, the actin-crosslinking activity of VgrG1 is essential to cause inflammatory diarrhea in infant mice and rabbits [28,47] (Figure 2a). In other pathogens, T6SSs have minor or no contributions to pathogenicity [48,49]. In some cases, inactivation of a T6SS even increases virulence, suggesting that T6SSs might limit acute pathogenic activities in these bacteria [35,50]. For some bacterial species even an inverse correlation between pathogenicity and T6SS abundance was noticed [51]. Many bacteria harboring a T6SS are not pathogens, indicating additional physiological functions of T6SSs unassociated with virulence. T6SSs have now
been demonstrated to provide an effective weaponry against Gram-negative bacteria and unicellular eukaryotes. They were originally identified by a genetic screen in V. cholerae to isolate factors required to protect the bacteria from predation by the unicellular eukaryote Dictyostelium discoideum [1]. An antibacterial activity for T6SSs was first reported for P. aeruginosa by Mougous and coworkers and was also subsequently demonstrated for T6SSs of V. cholerae, Burkholderia thailandensis and Serratia marcescens [7,8,52,53]. The T6SS of Pseudomonas syringae allows it to survive in presence of Saccharomyces cerevisiae by suppressing growth of yeast cells in competition experiments [54]. The dual role of T6SSs in pathogenicity and interbacterial competition is best exemplified by the specialized T6SSs of B. thailandensis, which encodes for five T6SSs. In this organism one T6SS (T6SS-1) is required for growth in the presence of competing bacteria, whereas another T6SS (T6SS-5) is essential for virulence in a mouse infection model [52]. In P. aeruginosa, the antibacterial activity is provided by the peptidoglycan degrading effectors Tse1 and Tse3, which are targeted to the periplasm of prey cells in an
( Figure 1 Legend Continued ) the ejection of the tail tube and the associated tail spike complex and results in puncturing of the outer membrane of the bacterial host. This ultimately results in the delivery of the viral DNA stored in the head. (b) Hcp tubes and an associated VgrG trimer exhibit structural similarity to the viral cell puncturing device. Hcp tubes are suggested to be engulfed by extended VipA/VipB tubules, which function in analogy to viral tail sheath proteins. The complex is anchored to the cell envelope via a membrane-associated complex, composed of TssJ, TssM (IcmF) and TssL (DotU), and a baseplate-like structure. Contraction of VipA/VipB causes ejection of Hcp and VgrG. Ejected Hcp and VgrG might dismantle from the cell surface. Contracted VipA/VipB tubules are subsequently removed by the ATPase ClpV, allowing for a new round of Hcp, VgrG, and VipA/VipB assembly and thereby a new round of secretion. OM: outer membrane; IM: inner membrane; PG: peptidoglycan layer. www.sciencedirect.com
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56 Host–microbe interactions: bacteria
H1-T6SS-dependent manner (Figure 2b) [6]. Tse1 and Tse3 exhibit different enzymatic activities, which may allow P. aeruginosa to kill a broader range of bacteria. The effector proteins are not linked to the T6SS-encoding gene cluster, but are coorganized with specific immunity proteins (e.g. Tse1), which protect P. aeruginosa cells from self killing by endogenous or intercellularly transferred toxins (Figure 2b) [6,8]. Using a heuristic approach 51 Tse1/Tsi1 toxin–antitoxin pairs were predicted in a broad range of proteobacterial species, all of them encoding for T6SSs [32]. A T6SS-dependent secretion of a predicted toxin family member and the presence of a periplasmic immunity protein for self-protection have been recently demonstrated for S. marcescens [55]. The predicted Tse1/ Tsi1 pairs were enriched in bacteria that face high densities of competing bacteria, for example, in soil or inside the gastrointestinal tract [32]. This suggests that T6SSs can increase bacterial fitness, allowing species that contain T6SSs to outcompete other bacteria. The antibacterial potential of T6SSs is also reflected by fusions of a peptidoglycan-binding domain or a LysM domain, showing structural homology to lysozyme, to V. cholerae or Pantoea agglomerans VgrGs [9,26]. Furthermore, S-type pyocins are fused to either S. enterica VgrG or uropathogenic E. coli Hcp, suggesting that the corresponding T6SSs deliver the bacteriocins to target cells upon membrane puncturing [56]. The advantageous effect of T6SSs in interbacterial competition may also have important supportive roles in polymicrobial infections. The human body harbors a complex and competitive microbiota and pathogens must displace commensal bacteria, which form a protective barrier preventing invader colonization. T6SSs can enable pathogens to kill off resident commensal bacteria, facilitating colonization of the host. This indirect role of T6SSs in virulence might have important impacts on virulence development and disease outcome [51]. T6SS activities are not only restricted to virulence toward eukaryotic and prokaryotic hosts but can also be involved in controlling host specificity [57] or biofilm formation [24,39]. T6SS components enable Proteus mirabilis cells to distinguish self from non-self [58]. The type VI secretion is a versatile toolbox, which enables Gram-negative bacteria to communicate with other cells in many contexts for diverse outputs.
Future directions It is now established that T6SSs form contractile machineries for membrane perforation and toxin delivery. Still, we are far from understanding the precise mechanism of T6S as many essential components remain poorly characterized. It is unclear whether classical effector are indeed passing an Hcp conduit and if so, how their transport is energized. Initial data indicate that T6SS activity can be regulated post-translationally allowing for the integration of environmental cues. The nature of these signals and Current Opinion in Microbiology 2013, 16:52–58
the signal transduction cascades are largely unknown. Is T6SS activation triggered by cell-to-cell contact? Does such regulation involve control of T6SS assembly or contraction of the VipA/VipB sheath structure? The dual role of T6SSs in pathogenicity and antibacterial virulence makes them an attractive target for muchneeded novel antibacterial therapeutics. Furthermore, surface-exposure of various T6SS components qualifies them as well-suited candidates for vaccination. The potential role of antibacterial T6SS activity in disease outcome is, however, still in need of documentation. Additional cellular targets of T6SSs in eukaryotic hosts remain to be identified. On the basis of bioinformatic analysis, potential functions of evolved VgrGs and classical effectors have been predicted, but remain to be confirmed. The rapid progress that has been achieved since the discovery of T6SSs holds great promise that these gaps can be filled soon.
Acknowledgements We thank Damon Huber, Gabriele Bo¨nemann and Lys Guilbride for editing of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A.M. (MO970/3). N.K. was supported by the Hartmut Hoffman-Berling International Graduate School of Molecular and Cellular Biology (HBIGS).
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