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Type VI Secretion System Helps Find a Niche
Cell Host & Microbe
Previews Type VI Secretion System Helps Find a Niche Nicole Kapitein1,2 and Axel Mogk1,2,* 1Zentrum fu ¨ r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany 2Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2014.06.012
Type VI secretion systems (T6SSs) deliver toxins into target cells and thus play a role in bacterial warfare. In this issue of Cell Host & Microbe, Ma et al. (2014) demonstrate that T6SS-dependent attack during interbacterial competition in the host context enables niche colonization by Agrobacterium tumefaciens. Competitive pressure rules the basic interactions between all organisms. The competition for nutrients and space essentially influences how microorganisms emerge and vanish in specific habitats. Bacteria extensively use two strategies to compete with each other. In a passive scramble competition, limiting resources are used up rapidly. The secretion of siderophores that efficiently sequester extracellular iron, preventing iron uptake by competing cells, exemplifies this deprivation of supplies. A more active contest competition involves direct interaction with competitors. The latter strategy encompasses the secretion into the extracellular space of small antimicrobial compounds including antibiotics or bacteriocins to poison opposing strains. A recently described specialized secretion machinery, the type VI secretion system (T6SS), is used by a wide variety of Gram-negative bacteria to directly deliver toxins upon cell-to-cell contact into target cells in order to kill. This cytotoxic activity of the T6SS was demonstrated to be targeted against both prokaryotic and eukaryotic competitors (Hood et al., 2010; Pukatzki et al., 2006). T6SS activity is not restricted to competitor cells but can also play important roles in pathogenicity in mammalian hosts. T6SSs have been extensively studied in the past years. They are typically encoded within a single gene cluster consisting of 13 conserved core components and a number of accessory ones. Together, those proteins form a membrane-embedded, syringe-like system strikingly similar to the injection machinery of bacteriophages. Analogous to the phage tail sheath proteins, the T6SS components VipA/VipB (TssB/
TssC) form a contractile sheath around a hollow, inner tube composed of Hcp (hemolysin-coregulated protein). Attached on top of the Hcp tube is a trimeric, spike-like cap consisting of VgrG (valine-glycine repeat protein G), similar to the tail spike complex of bacteriophages. Upon contraction of the VipA/ VipB (TssB/TssC) sheath, the Hcp tube together with VgrG is pushed outward and penetrates the target cell (for review see Kapitein and Mogk, 2013). The penetration of target cells is accompanied by the delivery of specific toxins. Effectors can be attached to the VgrG spike via specific PAAR domain-containing adaptors or are covalently fused to VgrG (Shneider et al., 2013). Alternatively, at least small toxins might be delivered directly through the hollow channel of the Hcp tube, ending up in the target cell after the VgrG cap has detached (Silverman et al., 2013). The number of T6SS-dependent effectors is manifold and mostly directed against highly conserved, indispensable cellular components: the peptidoglycan layer, membranes, and DNA. T6SS effectors co-occur in tandem with corresponding immunity proteins that inhibit the activity of the cognate toxin, preventing self-intoxication. The composition of the effector-immunity arsenal differs substantially even between closely related species, and the combination selected out of this big armory will determine the winners of interbacterial duels. T6SS function in interbacterial competition is also reflected in the control of its activity. In Pseudomonas aeruginosa, T6SS activity is controlled by the kinase PpkA, which is in turn regulated by the membrane-localized TagQRST protein complex. This membrane system senses
perturbations of the bacterial membranes, caused by an opponent cell, and transduces a yet-to-be-determined signal to ultimately trigger a T6SS counterstrike immediately after the initial attack (Basler et al., 2013). By now, multiple cases have been described where T6SS-positive organisms are able to efficiently outcompete competitor cells. This drives the hypothesis that T6SSs are important evolutionary factors helping bacteria to conquer ecological niches. Such a role could be also important for pathogens, enabling T6SS-positive populations to outcompete commensal bacteria, thereby indirectly supporting pathogenesis. While prior studies could demonstrate that T6SSs in Vibrio cholera are active upon colonization of the host intestinal tract, evidence for V. cholerae T6SS function in the replacement of the commensal microbiota is outstanding (Fu et al., 2013). Also, bacterial duels were so far not tested in physiological relevant environments, leaving the hypothesis, while attractive and reasonable, unproven. This open issue of the role of T6SS in interbacterial competition in situ has now been addressed by Ma et al. (2014). In their paper published in this issue of Cell Host & Microbe, Ma et al., (2014) used Agrobacterium tumefaciens, a Gram-negative soil bacterium that lives in the phyllosphere and causes the crown gall disease in infected plants, as the model T6SS expressing bacterium. Ma et al. (2014) show that A. tumefaciens T6SS is important for host colonization and involved both in intra- and interspecies competition with the T6SS elaborating soil bacterium Pseudomonas aeruginosa. When bacterial
Cell Host & Microbe 16, July 9, 2014 ª2014 Elsevier Inc. 5
Cell Host & Microbe
Previews A
B
Figure 1. The Natural Habitat Enables A. tumefaciens to Outcompete Opponent Bacteria via T6SS Activity (A and B) Bacterial duels between Agrobacterium tumefaciens and Pseudomonas aeruginosa. Pseudomonas aeruginosa efficiently outcompetes Agrobacterium tumefaciens in vitro (A), while the outcome of interbacterial competition is reversed in planta (B). Here, an unknown environmental signal presumably increases A. tumefaciens T6SS activity, leading to efficient delivery of Tde effectors that degrade chromosomal DNA of competitor cells. The immunity protein Tdi inactivates Tde and protects A. tumefaciens from self-toxication.
duels between A. tumefaciens and P. aeruginosa were performed in vitro on agar plates, A. tumefaciens was efficiently outcompeted (Figure 1). The competitive advantage of P. aeruginosa included a T6SS-mediated counterstrike. Strikingly, the outcome of the duel was reverted in planta, when coinfection experiments in tobacco plants were performed. Here, A. tumefaciens exhibited a T6SSmediated growth advantage toward P. aeruginosa, providing first evidence that T6SSs enable bacteria to settle in natural habitats and underlining the impact of the plant environment in this process. Competitive fitness of A. tumefaciens in planta is mediated by a novel class of T6SS-dependent DNase effectors, termed Tde (Figure 1). The Tde superfamily is likely VgrG linked and thereby delivered through association with the needle itself. Tde forms a classical toxin-antitoxin pair with the accompanying immunity protein Tdi. The authors confirm the widespread conservation of the Tde superfamily within Gram-negative plant pathogens and symbionts, thereby supporting a global role within plant colonization. The basis for the observed advantage enjoyed by A. tumefaciens within the plant habitat compared to an in vitro setting remains unclear. One possibility would
be that the A. tumefaciens T6SS activity is simply higher in planta, thereby leapfrogging Pseudomonas aeruginosa in its counterstrike activity. A. tumefaciens T6SSs can be controlled at transcriptional and posttranslational levels. Low pH increases expression of A. tumefaciens T6SS (Wu et al., 2012), but whether differences in acidity between in vitro and in planta experiments are causative of the opposing competition outcomes remains to be investigated. A. tumefaciens T6SS activity is also regulated by PpkAmediated phosphorylation, but TagQRST homologs are not present, leaving PpkA activity control unresolved (Lin et al., 2014). Therefore, further experimental advances aimed at identifying the habitat and host-specific signals that regulate the activation cascade of the A. tumefaciens T6SS would be required. The presented work sets the stage for deeper analysis of T6SSs in the natural environment and how their activities might be modulated by host factors. It will be important to broaden the now proven concept of T6SS function in niche occupation for other bacterial species. This understanding should also prove valuable for analyzing and understanding the contribution of T6SSs in acute and chronic diseases involving displacement of commensal bacteria.
6 Cell Host & Microbe 16, July 9, 2014 ª2014 Elsevier Inc.
ACKNOWLEDGMENTS This work was supported by grants of the German research council (DFG) to A.M. (MO970/3). REFERENCES Basler, M., Ho, B.T., and Mekalanos, J.J. (2013). Cell 152, 884–894. Fu, Y., Waldor, M.K., and Mekalanos, J.J. (2013). Cell Host Microbe 14, 652–663. Hood, R.D., Singh, P., Hsu, F., Gu¨vener, T., Carl, M.A., Trinidad, R.R., Silverman, J.M., Ohlson, B.B., Hicks, K.G., Plemel, R.L., et al. (2010). Cell Host Microbe 7, 25–37. Kapitein, N., and Mogk, A. (2013). Curr. Opin. Microbiol. 16, 52–58. Lin, J.S., Wu, H.H., Hsu, P.H., Ma, L.S., Pang, Y.Y., Tsai, M.D., and Lai, E.M. (2014). PLoS Pathog. 10, e1003991. Ma, L.-S., Hachani, A., Lin, J.-S., Filloux, A., and Lai, E.-M. (2014). Cell Host Microbe 16, this issue, 94–104. Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W.C., Heidelberg, J.F., and Mekalanos, J.J. (2006). Proc. Natl. Acad. Sci. USA 103, 1528–1533. Shneider, M.M., Buth, S.A., Ho, B.T., Basler, M., Mekalanos, J.J., and Leiman, P.G. (2013). Nature 500, 350–353. Silverman, J.M., Agnello, D.M., Zheng, H., Andrews, B.T., Li, M., Catalano, C.E., Gonen, T., and Mougous, J.D. (2013). Mol. Cell 51, 584–593. Wu, C.F., Lin, J.S., Shaw, G.C., and Lai, E.M. (2012). PLoS Pathog. 8, e1002938.
Previews Type VI Secretion System Helps Find a Niche Nicole Kapitein1,2 and Axel Mogk1,2,* 1Zentrum fu ¨ r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany 2Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2014.06.012
Type VI secretion systems (T6SSs) deliver toxins into target cells and thus play a role in bacterial warfare. In this issue of Cell Host & Microbe, Ma et al. (2014) demonstrate that T6SS-dependent attack during interbacterial competition in the host context enables niche colonization by Agrobacterium tumefaciens. Competitive pressure rules the basic interactions between all organisms. The competition for nutrients and space essentially influences how microorganisms emerge and vanish in specific habitats. Bacteria extensively use two strategies to compete with each other. In a passive scramble competition, limiting resources are used up rapidly. The secretion of siderophores that efficiently sequester extracellular iron, preventing iron uptake by competing cells, exemplifies this deprivation of supplies. A more active contest competition involves direct interaction with competitors. The latter strategy encompasses the secretion into the extracellular space of small antimicrobial compounds including antibiotics or bacteriocins to poison opposing strains. A recently described specialized secretion machinery, the type VI secretion system (T6SS), is used by a wide variety of Gram-negative bacteria to directly deliver toxins upon cell-to-cell contact into target cells in order to kill. This cytotoxic activity of the T6SS was demonstrated to be targeted against both prokaryotic and eukaryotic competitors (Hood et al., 2010; Pukatzki et al., 2006). T6SS activity is not restricted to competitor cells but can also play important roles in pathogenicity in mammalian hosts. T6SSs have been extensively studied in the past years. They are typically encoded within a single gene cluster consisting of 13 conserved core components and a number of accessory ones. Together, those proteins form a membrane-embedded, syringe-like system strikingly similar to the injection machinery of bacteriophages. Analogous to the phage tail sheath proteins, the T6SS components VipA/VipB (TssB/
TssC) form a contractile sheath around a hollow, inner tube composed of Hcp (hemolysin-coregulated protein). Attached on top of the Hcp tube is a trimeric, spike-like cap consisting of VgrG (valine-glycine repeat protein G), similar to the tail spike complex of bacteriophages. Upon contraction of the VipA/ VipB (TssB/TssC) sheath, the Hcp tube together with VgrG is pushed outward and penetrates the target cell (for review see Kapitein and Mogk, 2013). The penetration of target cells is accompanied by the delivery of specific toxins. Effectors can be attached to the VgrG spike via specific PAAR domain-containing adaptors or are covalently fused to VgrG (Shneider et al., 2013). Alternatively, at least small toxins might be delivered directly through the hollow channel of the Hcp tube, ending up in the target cell after the VgrG cap has detached (Silverman et al., 2013). The number of T6SS-dependent effectors is manifold and mostly directed against highly conserved, indispensable cellular components: the peptidoglycan layer, membranes, and DNA. T6SS effectors co-occur in tandem with corresponding immunity proteins that inhibit the activity of the cognate toxin, preventing self-intoxication. The composition of the effector-immunity arsenal differs substantially even between closely related species, and the combination selected out of this big armory will determine the winners of interbacterial duels. T6SS function in interbacterial competition is also reflected in the control of its activity. In Pseudomonas aeruginosa, T6SS activity is controlled by the kinase PpkA, which is in turn regulated by the membrane-localized TagQRST protein complex. This membrane system senses
perturbations of the bacterial membranes, caused by an opponent cell, and transduces a yet-to-be-determined signal to ultimately trigger a T6SS counterstrike immediately after the initial attack (Basler et al., 2013). By now, multiple cases have been described where T6SS-positive organisms are able to efficiently outcompete competitor cells. This drives the hypothesis that T6SSs are important evolutionary factors helping bacteria to conquer ecological niches. Such a role could be also important for pathogens, enabling T6SS-positive populations to outcompete commensal bacteria, thereby indirectly supporting pathogenesis. While prior studies could demonstrate that T6SSs in Vibrio cholera are active upon colonization of the host intestinal tract, evidence for V. cholerae T6SS function in the replacement of the commensal microbiota is outstanding (Fu et al., 2013). Also, bacterial duels were so far not tested in physiological relevant environments, leaving the hypothesis, while attractive and reasonable, unproven. This open issue of the role of T6SS in interbacterial competition in situ has now been addressed by Ma et al. (2014). In their paper published in this issue of Cell Host & Microbe, Ma et al., (2014) used Agrobacterium tumefaciens, a Gram-negative soil bacterium that lives in the phyllosphere and causes the crown gall disease in infected plants, as the model T6SS expressing bacterium. Ma et al. (2014) show that A. tumefaciens T6SS is important for host colonization and involved both in intra- and interspecies competition with the T6SS elaborating soil bacterium Pseudomonas aeruginosa. When bacterial
Cell Host & Microbe 16, July 9, 2014 ª2014 Elsevier Inc. 5
Cell Host & Microbe
Previews A
B
Figure 1. The Natural Habitat Enables A. tumefaciens to Outcompete Opponent Bacteria via T6SS Activity (A and B) Bacterial duels between Agrobacterium tumefaciens and Pseudomonas aeruginosa. Pseudomonas aeruginosa efficiently outcompetes Agrobacterium tumefaciens in vitro (A), while the outcome of interbacterial competition is reversed in planta (B). Here, an unknown environmental signal presumably increases A. tumefaciens T6SS activity, leading to efficient delivery of Tde effectors that degrade chromosomal DNA of competitor cells. The immunity protein Tdi inactivates Tde and protects A. tumefaciens from self-toxication.
duels between A. tumefaciens and P. aeruginosa were performed in vitro on agar plates, A. tumefaciens was efficiently outcompeted (Figure 1). The competitive advantage of P. aeruginosa included a T6SS-mediated counterstrike. Strikingly, the outcome of the duel was reverted in planta, when coinfection experiments in tobacco plants were performed. Here, A. tumefaciens exhibited a T6SSmediated growth advantage toward P. aeruginosa, providing first evidence that T6SSs enable bacteria to settle in natural habitats and underlining the impact of the plant environment in this process. Competitive fitness of A. tumefaciens in planta is mediated by a novel class of T6SS-dependent DNase effectors, termed Tde (Figure 1). The Tde superfamily is likely VgrG linked and thereby delivered through association with the needle itself. Tde forms a classical toxin-antitoxin pair with the accompanying immunity protein Tdi. The authors confirm the widespread conservation of the Tde superfamily within Gram-negative plant pathogens and symbionts, thereby supporting a global role within plant colonization. The basis for the observed advantage enjoyed by A. tumefaciens within the plant habitat compared to an in vitro setting remains unclear. One possibility would
be that the A. tumefaciens T6SS activity is simply higher in planta, thereby leapfrogging Pseudomonas aeruginosa in its counterstrike activity. A. tumefaciens T6SSs can be controlled at transcriptional and posttranslational levels. Low pH increases expression of A. tumefaciens T6SS (Wu et al., 2012), but whether differences in acidity between in vitro and in planta experiments are causative of the opposing competition outcomes remains to be investigated. A. tumefaciens T6SS activity is also regulated by PpkAmediated phosphorylation, but TagQRST homologs are not present, leaving PpkA activity control unresolved (Lin et al., 2014). Therefore, further experimental advances aimed at identifying the habitat and host-specific signals that regulate the activation cascade of the A. tumefaciens T6SS would be required. The presented work sets the stage for deeper analysis of T6SSs in the natural environment and how their activities might be modulated by host factors. It will be important to broaden the now proven concept of T6SS function in niche occupation for other bacterial species. This understanding should also prove valuable for analyzing and understanding the contribution of T6SSs in acute and chronic diseases involving displacement of commensal bacteria.
6 Cell Host & Microbe 16, July 9, 2014 ª2014 Elsevier Inc.
ACKNOWLEDGMENTS This work was supported by grants of the German research council (DFG) to A.M. (MO970/3). REFERENCES Basler, M., Ho, B.T., and Mekalanos, J.J. (2013). Cell 152, 884–894. Fu, Y., Waldor, M.K., and Mekalanos, J.J. (2013). Cell Host Microbe 14, 652–663. Hood, R.D., Singh, P., Hsu, F., Gu¨vener, T., Carl, M.A., Trinidad, R.R., Silverman, J.M., Ohlson, B.B., Hicks, K.G., Plemel, R.L., et al. (2010). Cell Host Microbe 7, 25–37. Kapitein, N., and Mogk, A. (2013). Curr. Opin. Microbiol. 16, 52–58. Lin, J.S., Wu, H.H., Hsu, P.H., Ma, L.S., Pang, Y.Y., Tsai, M.D., and Lai, E.M. (2014). PLoS Pathog. 10, e1003991. Ma, L.-S., Hachani, A., Lin, J.-S., Filloux, A., and Lai, E.-M. (2014). Cell Host Microbe 16, this issue, 94–104. Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W.C., Heidelberg, J.F., and Mekalanos, J.J. (2006). Proc. Natl. Acad. Sci. USA 103, 1528–1533. Shneider, M.M., Buth, S.A., Ho, B.T., Basler, M., Mekalanos, J.J., and Leiman, P.G. (2013). Nature 500, 350–353. Silverman, J.M., Agnello, D.M., Zheng, H., Andrews, B.T., Li, M., Catalano, C.E., Gonen, T., and Mougous, J.D. (2013). Mol. Cell 51, 584–593. Wu, C.F., Lin, J.S., Shaw, G.C., and Lai, E.M. (2012). PLoS Pathog. 8, e1002938.