TIMI-1091; No. of Pages 3
Spotlight
The target cell genus does not matter Sophie Bleves, Thibault G. Sana, and Rome´ Voulhoux CNRS & Aix-Marseille Universite´, Laboratoire d’Inge´nierie des Syste`mes Macromole´culaires (UMR7255), Institut de Microbiologie de la Me´diterrane´e (IMM), Marseille, France
Two type VI secreted phospholipases D of Pseudomonas aeruginosa were identified as trans-kingdom virulence effectors, targeting both prokaryotic and eukaryotic host cells. Each of them triggers killing bacterial competitors and internalization into non-phagocytic cells. These type VI lipolytic enzymes are widely distributed among pathogens and may constitute a conserved strategy. Initially identified for a role in pathogenesis against eukaryotic host cells, the major physiological significance of the type VI secretion system (T6SS) is to provide defense against other bacteria in the environment [1,2]. T6SSs are often found in several copies in a genome, with some being dedicated to interaction with prokaryotes while others with eukaryotes, as occurs in Burkholderia for example. In the case of a bacterium like Vibrio cholerae, which harbors a single T6SS known to mediate interactions with both type of hosts, the nature of the effector determines the effect: the effector VgrG1 acts through an actin crosslinking domain to modify the host cytoskeleton, while the muramidase VgrG3 attacks the bacterial cell wall [1,2]. According to a recent report from Jiang et al. [3], a novel class of T6SS effectors that targets both prokaryotic and eukaryotic host cells have been identified in Pseudomonas aeruginosa. The T6SS-secreted phospholipases D PldA and PldB are thus the first examples of trans-kingdom effectors. P. aeruginosa is one of the leading causes of nosocomial infections in immunocompromised humans and is responsible for chronic respiratory disease of patients with cystic fibrosis. The pathogen harbors three distinct T6SS gene loci. The H1-T6SS delivers at least 6 toxic effectors into host bacteria and is a model for studying physiological function of T6SS antimicrobial activity (Figure 1) [1,2,4]. It may allow P. aeruginosa to more efficiently compete with other bacteria in the environment, or in the context of the infection to defend a niche. The H2-T6SS has a dual role allowing interaction with both eukaryotic and prokaryotic target cells (Figure 1). H2-T6SS contributes, on the one hand, to P. aeruginosa immune evasion by promoting internalization into non-phagocytic cells through a yet unidentified effector [5], and, on the other hand, it mediates antibacterial activity through the phospholipase D (PLD) PldA [6]. PldA, also named Tle5, belongs to a diverse superfamily of lipases, called type VI lipase effectors (Tle), that has been recently shown to mediate potent antimicrobial activity. Little was known about the function of the Corresponding author: Bleves, S. ([email protected]). 0966-842X/ ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tim.2014.04.011
third system (H3-T6SS) before the report of Jiang and colleagues [3], except that it was transcriptionally connected to H2-T6SS during host infection [7,8]. P. aeruginosa strain PAO1 contains a second T6SSassociated PLD, called PldB, and the authors hypothesized that it could function as a bacterial toxin, similar to PldA [6]. Indeed production of PldB in the periplasm of Escherichia coli inhibits its growth. Consistent with this, a histidine catalytic mutant form of PldB had no effect, demonstrating that PldB acts as a PLD antibacterial effector. Interestingly a connection between PldB and the H3-T6SS was suggested as the pldB expression pattern follows the induction and maximal expression of the H3T6SS in late stationary phase at 37 8C [8]. Accordingly, in these specific conditions, only the inactivation of H3-T6SS or PldB abolished toxicity in intrabacterial as well in interbacterial competition assays. The products of the three genes downstream of pldB were shown to function as immunity proteins against PldB toxicity. Indeed, in order to protect themselves from self or sister cell intoxication, bacteria produce immunity proteins to neutralize their cognate T6SS antibacterial effector (Figure 1). Given that in addition to a PldA mediated-antibacterial function [6], H2-T6SS contributes to invasion of nonphagocytic cells [5], Jiang and colleagues asked whether H3-T6SS and PldB could play a similar role. In H2-T6SS invasion conditions (i.e., bacteria grown in exponential phase), neither H3-T6SS nor PldB mutants participate in P. aeruginosa uptake by epithelial HeLa cells. Interestingly, in H3-T6SS conditions (i.e., bacteria grown at stationary phase), both of the T6SSs and both of the PLDs contribute to internalization, compensating for each other, in agreement with what was previously observed in acute mouse models of virulence [7]. The integrity of the PLD active sites of PldA and PldB is crucial for their role in pathogenicity. Moreover, PldA, PldB, and H3-T6SS are dispensable for cytotoxicity towards host cells, adherence and intracellular survival, and thus these likely only play a role in the entry step as demonstrated for H2-T6SS [5]. However, PldA contribution to the H2-T6SS mediated entry of exponential phase bacteria still has to be shown. The dependence on H2-T6SS for PldA and on H3-T6SS for PldB was further characterized by demonstrating their delivery in host cell cytosol upon infection. One can note that they are the first non-VgrG anti-eukaryotic effectors shown to be translocated into eukaryotic host cell. Once injected, PldA and PldB therefore could target a host component. Previous works have shown that the host PI3K/Akt signaling pathway is recruited and activated during P. aeruginosa internalization and mediated by H2-T6SS [5,9]. Jiang and colleagues showed that those Trends in Microbiology xx (2014) 1–3
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Spotlight
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
PI3K/Akt-dependent bacterial internalizaon
Eukaryoc target cell Cytoplasm Plasma mem brane
?
PldA PldB
P. aeruginosa H2
Cytop lasm Inner mem bran e Perip lasm
Outer
mem
Tsi Tse
PldA
H1
H2
Tsi
brane
PldB
H3 Tli5
1 5
H3
3
l1
PldA
l2
l3
PldB
4 6 2
Bacterial target cell TRENDS in Microbiology
Figure 1. Pseudomonas aeruginosa possesses three functional type VI secretion systems (T6SSs). While the H1-T6SS (H1) mediates interaction with bacteria through at least six different type VI exported (Tse) effectors (1–6), the H2 and H3-T6SS (H2 and H3) are involved in interaction with both bacterial and eukaryotic cells through the PldA and PldB trans-kingdom effectors. The immunity proteins (Tsi, Tli5, I1, I2, and I3) that protect the secreting cells are located in the periplasm or cytoplasm depending on the final destination of their cognate effector.
host kinases are also required for the H3-T6SS and PldBdependent internalization. They further showed that similar to a secreted PLD of Neisseria gonorrhoeae [10], PldA and PldB bind the Akt kinase. This direct interaction may lead to Akt activation, which results in actin-based membrane extensions triggering P. aeruginosa internalization at the apical face of the epithelial cell. The findings of Jiang and colleagues [3] provide new and important insights into the pathogenesis of P. aeruginosa (Figure 1). PldB is thus the first identified effector for the H3-T6SS. Its PLD activity is toxic to other bacteria and is neutralized by three different immunity proteins. It is also so far the first case of a single antibacterial toxin inhibited by several immunity proteins. What is even more interesting with this effector is its implication during the interaction with both prokaryotic and eukaryotic host cells. PldB is the paradigm of a trans-kingdom effector, and PldA appears to have a similar function. If not restricted to P. aeruginosa, and this class of lipolytic enzymes are found in diverse pathogens including Vibrio and Burkholderia, then this strategy of ‘one effector for any type of target cells’ is 2
very simple, but terribly effective. The target cell genus does not matter. Acknowledgments The authors thank members of Rome´ Voulhoux’s team and Mr. Amsterdam for their constant support, and acknowledge funding through the CNRS and Aix-Marseille Universite´, Vaincre La Mucoviscidose grants (10IC1002, RF20120600685, RF20130500911) and ‘Pathomics’ ERA-net PATHO Grant ANR-08-PATH-004-01.
References 1 Ho, B.T. et al. (2014) A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15, 9–21 2 Russel, A.B. et al. (2014) Type VI secretion system effectors: poisons with a purpose. Nat. Rev. Microbiol. 12, 137–148 3 Jiang, F. et al. (2014) A single Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe http://dx.doi.org/10.1016/ j.chom.2014.04.010 4 Whitney, J.C. et al. (2014) Genetically distinct pathways guide effector export through the type VI secretion system. Mol. Microbiol. 92, 529–542 5 Sana, T.G. et al. (2012) The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum
TIMI-1091; No. of Pages 3
Spotlight sensing and Fur and modulates internalization in epithelial cells. J. Biol. Chem. 287, 27095–27105 6 Russel, A.B. et al. (2013) Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 496, 508–512 7 Lesic, B. et al. (2009) Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis. Microbiology 155, 2845–2855
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
8 Sana, T.G. et al. (2013) Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa. PLoS ONE 8, e76030 9 Kierbel, A. et al. (2005) The phosphoinositol-3-kinase-protein kinase B/ Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization. Mol. Biol. Cell 16, 2577–2585 10 Edwards, J.L. and Apicella, M.A. (2006) Neisseria gonorrhoeae PLD directly interacts with Akt kinase upon infection of primary, human, cervical epithelial cells. Cell. Microbiol. 8, 1253–1271
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Spotlight
The target cell genus does not matter Sophie Bleves, Thibault G. Sana, and Rome´ Voulhoux CNRS & Aix-Marseille Universite´, Laboratoire d’Inge´nierie des Syste`mes Macromole´culaires (UMR7255), Institut de Microbiologie de la Me´diterrane´e (IMM), Marseille, France
Two type VI secreted phospholipases D of Pseudomonas aeruginosa were identified as trans-kingdom virulence effectors, targeting both prokaryotic and eukaryotic host cells. Each of them triggers killing bacterial competitors and internalization into non-phagocytic cells. These type VI lipolytic enzymes are widely distributed among pathogens and may constitute a conserved strategy. Initially identified for a role in pathogenesis against eukaryotic host cells, the major physiological significance of the type VI secretion system (T6SS) is to provide defense against other bacteria in the environment [1,2]. T6SSs are often found in several copies in a genome, with some being dedicated to interaction with prokaryotes while others with eukaryotes, as occurs in Burkholderia for example. In the case of a bacterium like Vibrio cholerae, which harbors a single T6SS known to mediate interactions with both type of hosts, the nature of the effector determines the effect: the effector VgrG1 acts through an actin crosslinking domain to modify the host cytoskeleton, while the muramidase VgrG3 attacks the bacterial cell wall [1,2]. According to a recent report from Jiang et al. [3], a novel class of T6SS effectors that targets both prokaryotic and eukaryotic host cells have been identified in Pseudomonas aeruginosa. The T6SS-secreted phospholipases D PldA and PldB are thus the first examples of trans-kingdom effectors. P. aeruginosa is one of the leading causes of nosocomial infections in immunocompromised humans and is responsible for chronic respiratory disease of patients with cystic fibrosis. The pathogen harbors three distinct T6SS gene loci. The H1-T6SS delivers at least 6 toxic effectors into host bacteria and is a model for studying physiological function of T6SS antimicrobial activity (Figure 1) [1,2,4]. It may allow P. aeruginosa to more efficiently compete with other bacteria in the environment, or in the context of the infection to defend a niche. The H2-T6SS has a dual role allowing interaction with both eukaryotic and prokaryotic target cells (Figure 1). H2-T6SS contributes, on the one hand, to P. aeruginosa immune evasion by promoting internalization into non-phagocytic cells through a yet unidentified effector [5], and, on the other hand, it mediates antibacterial activity through the phospholipase D (PLD) PldA [6]. PldA, also named Tle5, belongs to a diverse superfamily of lipases, called type VI lipase effectors (Tle), that has been recently shown to mediate potent antimicrobial activity. Little was known about the function of the Corresponding author: Bleves, S. ([email protected]). 0966-842X/ ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tim.2014.04.011
third system (H3-T6SS) before the report of Jiang and colleagues [3], except that it was transcriptionally connected to H2-T6SS during host infection [7,8]. P. aeruginosa strain PAO1 contains a second T6SSassociated PLD, called PldB, and the authors hypothesized that it could function as a bacterial toxin, similar to PldA [6]. Indeed production of PldB in the periplasm of Escherichia coli inhibits its growth. Consistent with this, a histidine catalytic mutant form of PldB had no effect, demonstrating that PldB acts as a PLD antibacterial effector. Interestingly a connection between PldB and the H3-T6SS was suggested as the pldB expression pattern follows the induction and maximal expression of the H3T6SS in late stationary phase at 37 8C [8]. Accordingly, in these specific conditions, only the inactivation of H3-T6SS or PldB abolished toxicity in intrabacterial as well in interbacterial competition assays. The products of the three genes downstream of pldB were shown to function as immunity proteins against PldB toxicity. Indeed, in order to protect themselves from self or sister cell intoxication, bacteria produce immunity proteins to neutralize their cognate T6SS antibacterial effector (Figure 1). Given that in addition to a PldA mediated-antibacterial function [6], H2-T6SS contributes to invasion of nonphagocytic cells [5], Jiang and colleagues asked whether H3-T6SS and PldB could play a similar role. In H2-T6SS invasion conditions (i.e., bacteria grown in exponential phase), neither H3-T6SS nor PldB mutants participate in P. aeruginosa uptake by epithelial HeLa cells. Interestingly, in H3-T6SS conditions (i.e., bacteria grown at stationary phase), both of the T6SSs and both of the PLDs contribute to internalization, compensating for each other, in agreement with what was previously observed in acute mouse models of virulence [7]. The integrity of the PLD active sites of PldA and PldB is crucial for their role in pathogenicity. Moreover, PldA, PldB, and H3-T6SS are dispensable for cytotoxicity towards host cells, adherence and intracellular survival, and thus these likely only play a role in the entry step as demonstrated for H2-T6SS [5]. However, PldA contribution to the H2-T6SS mediated entry of exponential phase bacteria still has to be shown. The dependence on H2-T6SS for PldA and on H3-T6SS for PldB was further characterized by demonstrating their delivery in host cell cytosol upon infection. One can note that they are the first non-VgrG anti-eukaryotic effectors shown to be translocated into eukaryotic host cell. Once injected, PldA and PldB therefore could target a host component. Previous works have shown that the host PI3K/Akt signaling pathway is recruited and activated during P. aeruginosa internalization and mediated by H2-T6SS [5,9]. Jiang and colleagues showed that those Trends in Microbiology xx (2014) 1–3
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Spotlight
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
PI3K/Akt-dependent bacterial internalizaon
Eukaryoc target cell Cytoplasm Plasma mem brane
?
PldA PldB
P. aeruginosa H2
Cytop lasm Inner mem bran e Perip lasm
Outer
mem
Tsi Tse
PldA
H1
H2
Tsi
brane
PldB
H3 Tli5
1 5
H3
3
l1
PldA
l2
l3
PldB
4 6 2
Bacterial target cell TRENDS in Microbiology
Figure 1. Pseudomonas aeruginosa possesses three functional type VI secretion systems (T6SSs). While the H1-T6SS (H1) mediates interaction with bacteria through at least six different type VI exported (Tse) effectors (1–6), the H2 and H3-T6SS (H2 and H3) are involved in interaction with both bacterial and eukaryotic cells through the PldA and PldB trans-kingdom effectors. The immunity proteins (Tsi, Tli5, I1, I2, and I3) that protect the secreting cells are located in the periplasm or cytoplasm depending on the final destination of their cognate effector.
host kinases are also required for the H3-T6SS and PldBdependent internalization. They further showed that similar to a secreted PLD of Neisseria gonorrhoeae [10], PldA and PldB bind the Akt kinase. This direct interaction may lead to Akt activation, which results in actin-based membrane extensions triggering P. aeruginosa internalization at the apical face of the epithelial cell. The findings of Jiang and colleagues [3] provide new and important insights into the pathogenesis of P. aeruginosa (Figure 1). PldB is thus the first identified effector for the H3-T6SS. Its PLD activity is toxic to other bacteria and is neutralized by three different immunity proteins. It is also so far the first case of a single antibacterial toxin inhibited by several immunity proteins. What is even more interesting with this effector is its implication during the interaction with both prokaryotic and eukaryotic host cells. PldB is the paradigm of a trans-kingdom effector, and PldA appears to have a similar function. If not restricted to P. aeruginosa, and this class of lipolytic enzymes are found in diverse pathogens including Vibrio and Burkholderia, then this strategy of ‘one effector for any type of target cells’ is 2
very simple, but terribly effective. The target cell genus does not matter. Acknowledgments The authors thank members of Rome´ Voulhoux’s team and Mr. Amsterdam for their constant support, and acknowledge funding through the CNRS and Aix-Marseille Universite´, Vaincre La Mucoviscidose grants (10IC1002, RF20120600685, RF20130500911) and ‘Pathomics’ ERA-net PATHO Grant ANR-08-PATH-004-01.
References 1 Ho, B.T. et al. (2014) A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15, 9–21 2 Russel, A.B. et al. (2014) Type VI secretion system effectors: poisons with a purpose. Nat. Rev. Microbiol. 12, 137–148 3 Jiang, F. et al. (2014) A single Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe http://dx.doi.org/10.1016/ j.chom.2014.04.010 4 Whitney, J.C. et al. (2014) Genetically distinct pathways guide effector export through the type VI secretion system. Mol. Microbiol. 92, 529–542 5 Sana, T.G. et al. (2012) The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum
TIMI-1091; No. of Pages 3
Spotlight sensing and Fur and modulates internalization in epithelial cells. J. Biol. Chem. 287, 27095–27105 6 Russel, A.B. et al. (2013) Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 496, 508–512 7 Lesic, B. et al. (2009) Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis. Microbiology 155, 2845–2855
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
8 Sana, T.G. et al. (2013) Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa. PLoS ONE 8, e76030 9 Kierbel, A. et al. (2005) The phosphoinositol-3-kinase-protein kinase B/ Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization. Mol. Biol. Cell 16, 2577–2585 10 Edwards, J.L. and Apicella, M.A. (2006) Neisseria gonorrhoeae PLD directly interacts with Akt kinase upon infection of primary, human, cervical epithelial cells. Cell. Microbiol. 8, 1253–1271
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