Microbiological Research 172 (2015) 19–25

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Review

Composition, function, and regulation of T6SS in Pseudomonas aeruginosa Lihua Chen, Yaru Zou, Pengfei She, Yong Wu ∗ Department of Medicine Clinical Laboratory, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan, PR China

a r t i c l e

i n f o

Article history: Received 1 November 2014 Received in revised form 3 January 2015 Accepted 3 January 2015 Available online 7 January 2015 Keywords: Pseudomonas aeruginosa Type VI secretion system Composition Function Regulation

a b s t r a c t Bacterial cells can communicate with their surrounding environment through secretion systems. Type VI secretion system (T6SS) is one of the most recently discovered secretion systems, which is distributed widely in Gram-negative bacteria such as Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen. This protein secretion system shares similarity with the puncturing device of bacteriophages in structure. P. aeruginosa is an important opportunistic pathogen and distributes widely in diverse environment. T6SS is beneficial to survival advantage of P. aeruginosa by delivering toxins to its neighboring pathogens and translocating protein effectors into the host cells. T6SS is also the virulence factor and takes part in biofilm formation of P. aeruginosa. The functions of T6SS in P. aeruginosa are regulated at transcriptional, posttranscriptional and posttranslational levels by diverse mechanisms. This article reviews the latest progress in the structure, effector proteins, biological function, and regulation mechanisms of P. aeruginosa T6SS. © 2015 Elsevier GmbH. All rights reserved.

Contents 1. 2. 3. 4.

5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Components of T6SS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effectors of P. aeruginosa T6SS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of P. aeruginosa T6SS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Delivery of toxin to other competing bacterial species to help the growth of P. aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. As the virulence factor of P. aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Takes part in bacterial biofilm formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of P. aeruginosa T6SS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Transcriptional regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Posttranscriptional regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Posttranslational regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. ␴54 factor (RpoN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Bacterial cells have developed multiple strategies to communicate with their surrounding environment. One of these strategies

∗ Corresponding author. Tel.: +86 731 88618441. E-mail address: wuyong [email protected] (Y. Wu). http://dx.doi.org/10.1016/j.micres.2015.01.004 0944-5013/© 2015 Elsevier GmbH. All rights reserved.

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is the secretion system. Using the secretion system, bacterial cells can obtain the nutrient, secrete toxic proteins to the outer environment, or directly act on targets of eukaryote resulting in illness. To date, seven types of secretion system have been found, namely type I secretion system to type VII secretion system (T1SS–T7SS). These secretion systems deliver extracellular proteins or effectors through secretion or injection, thus regulating the interaction between bacteria and host. The type VI secretion system (T6SS) is

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recognized as one of the most recent examples of these organelles. It was first defined in 2006 by two articles. The first study by Pukatzki et al. reported that secretion of the proteins hemolysin coregulated protein (Hcp) and valine–glycine repeat protein G (VgrG) in Vibrio cholerae required IcmF-associated homologous protein gene cluster, which was also necessary for cytotoxicity of V. cholerae toward Dictyostelium amoebae (Pukatzki et al. 2006). The second study published by Mougous et al. included structural data of a secretion apparatus, and provided evidence that the apparatus was functional in patients with cystic fibrosis with chronic lung infection due to Pseudomonas aeruginosa (P. aeruginosa) (Mougous et al., 2006). The initial studies on T6SS were focused on the role of T6SS in pathogenesis (Bröms et al., 2012; Burtnick et al., 2011; Suarez et al., 2008). However, recent studies showed that T6SS could also promote commensal or mutualistic relationships between bacteria and eukaryotic host, and mediate the function of cooperation or competition in interbacterial interactions (Decoin et al., 2014; Jani and Cotter 2010; Tashiro et al., 2013). P. aeruginosa is a kind of opportunistic Gram-negative pathogen and distributes widely in environment. It is responsible for a wide range of human diseases, such as septicaemia, pneumonia, and several other kinds of infection. This bacterium is the most often found pathogen leading to the respiratory tract of cystic fibrosis (CF). It encodes nearly all secretion systems described thus far, including three evolutionarily distinct T6SS clusters, which makes P. aeruginosa as one ideal model organism to study T6SS (Bleves et al., 2010; Filloux et al., 2008). The majority of recent T6SS studies have been focused on this bacteria genera. Therefore, in the present review we concentrate on P. aeruginosa. This review discusses the structure, function, and mechanisms of regulation of the P. aeruginosa T6SS to provide new insights for the control of infection caused by P. aeruginosa.

2. Components of T6SS Nearly 25% of all sequenced Gram-negative bacteria have the gene clusters encoding T6SS including V. cholerae, P. aeruginosa, and Acinetobacter. Genetic clusters of T6SS comprise a group of tightly clustered genes; among these 13 conserved genes are the core components (Boyer et al., 2009; Zheng and Leung 2007). Based on bioinformatic approaches, these genes can be classified into three categories (Silverman et al., 2012). The first category: genes encoding membrane-associated proteins such as integral membrane TssL, TssM or lipoproteins TssJ (named for type six subunit) (Zoued et al., 2013). They interact to form a complex that crosses the cell envelope. The second category: genes encoding proteins in relation to tailed bacteriophage components including: (1) the syringe of tailed bacteriophage: Hcp and VgrG. Hcp protein could ˚ It is similar in form tubes as hexamers with a diameter of 40 A. appearance to the major tail protein of phage , gpV. VgrG protein could form homotrimeric complexes and is related to the gp27/gp5 complex, the spike of bacteriophage T4 (Leiman et al., 2009); (2) the components similar to that of the sheath of the bacteriophage: TssB/TssC subunits (also called VipA/B components in V. cholerae). TssB and TssC could form tubular structures and were highly similar to gp18. TssB/TssC subunits could contract and provide energy to eject the tube protein-like Hcp structure, thereby possibly puncturing the target cell and injecting pathogenic effectors into its interior (Bönemann et al., 2010); (3) the components were similar to the bacteriophage baseplate: TssE. TssE is located in the cytoplasm and is related to the T4 phage baseplate gp25; (4) the components that provide energy: ClpV. ClpV has the characteristic of AAA+ ATPase. The contracted TssB/TssC is recognized by ClpV and is disintegrated by a process dependent on ClpV-mediated ATP hydrolysis (Records 2011) and (5) membrane-associated protein structure IcmF: IcmF

is an intermembrane protein with ATPase activity. It binds to IcmH and forms the transmembrane complex. IcmF is required for the structural components of T6SS and is the possible mosaic comprising bacteriophage tail tube. The third category includes the components with unidentified function such as TssA, TssF, TssG, and TssK. Fig. 1 shows the structural models of bacteriophage and T6SS (Ho et al., 2014); the homologous components between them are marked with the same color. In 2003, Das and Chaudhuri first demonstrated a gene cluster containing 15 genes surrounding IcmF and named it as IcmF associated homologous protein (IAHP) through in silico analysis. P. aeruginosa was the only bacteria genera that had three different IAHP/T6SS clusters among the nine identified gram-negative proteo-bacteria (Das and Chaudhuri 2003). In 2006, one of these clusters, named Hcp1 secretion island I (HSI-I), was first demonstrate by Mougous et al. in Science as a virulence locus of P. aeruginosa encoding a protein secretion apparatus. The secreted protein was Hcp1, a hexameric protein with a 40 A˚ internal diameter, which secretion was HSI-I-dependent and facilitated by ClpV1 (Mougous et al., 2006). The HSI-I-encoded T6SS of P. aeruginosa (H1-T6SS) exports three toxin proteins Tse1-3 (type six exported 1–3) to other bacteria and provides major fitness advantage for P. aeruginosa (Hood et al., 2010). In contrast to H1-T6SS targeting prokaryotic cells, H2-T6SS modulates internalization in epithelial cells through PI3K-Akt host pathway activation (Sana et al., 2012). Although the function of HSI-I and HSI-II have been partially studied, the knowledge on the homologous locus HSI-III is very limited. Only one report showed that HSI-III was import for Arabidopsis thaliana infection model and acute lung infection model (Lesic et al., 2009). In a recently phylogenetic analysis within the T6SS tree, three T6SSs in P. aeruginosa are found in different groups. For example, HSI-I belongs to group A, while HSI-II is in group B and HSI-III is in group D (Bingle et al., 2008). The three P. aeruginosa T6SSs have distinct evolutionary histories suggests that they have been acquired by horizontal transfer. The gene clusters of P. aeruginosa T6SSs are shown in Fig. 2 (Filloux et al., 2008). Table 1 shows the characteristics of genes involved.

3. Effectors of P. aeruginosa T6SS Effectors are the proteins secreted by the secretion system. The effectors ensure that the bacteria can interact with other bacteria and environment. With the discovery of P. aeruginosa T6SS, diverse effectors were identified. The first identified effector is the previously stated protein VgrG (Pukatzki et al., 2007). Hcp is not only a secreted protein itself by T6SS, it is also a chaperone and receptor of T6S effectors (Silverman et al., 2013). In 2010, Hood et al. identified that H1-T6SS of P. aeruginosa dilivers three effector proteins, namely Tse1-3 (type VI secretion exported 1–3) (Hood et al., 2010). Among these, Tse1 and Tse3 could degrade the peptidoglycan of the bacteria envelope dissolving the target cells. The crystal structure of Tse1 diffracts at a 1.5 A˚ resolution, including six ␤-strands (␤1–␤6) and five helices (␣1–␣5). It belongs to the superfamily of N1pC/P60 peptidases (Benz et al., 2012; Ding et al., 2012). Tse2 is the toxic component of the toxic immunity system, specialized in targeting the bacterial cytoplasm. Tse2 could inhibit the growth of other bacteria and provide a pronounced fitness advantage for P. aeruginosa (Li et al., 2012). Tse3 belongs to space group C121 and diffracts to 1.5 A˚ resolution (Lu et al., 2013). To avoid being killed by Tse1-3, P. aeruginosa have three immunity proteins namely Tsi1-3 (Tse1-3 specific immunity protein), which could block the activity of Tse1-3 (Hood et al., 2010; Russell et al., 2011). For example, the HI loop from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1 and interact with Tse1, thus blocking the binding of enzyme to

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Fig. 1. Structural model of bacteriophage and T6SS. Homologues and related genes are indicated with the same color. TM, target membrane; OM, outer membrane; IM, inner membrane (Ho et al., 2014).

peptidoglycan (Shang et al., 2012). In this way, P. aeruginosa can control the function of Tse1 and protect itself. The functional spectrum of a secretion system is defined by its substrates. Russell et al. showed that in P. aeruginosa, PldA, a eukaryotic-like phospholipase D (PLD), was also the lipase effector of T6SS (Russell et al., 2013). It was the substrate of H2-T6SS and a member of Tle5 family. PldA could degrade

phosphatidylethanolamine of the bacterial membrane and exert its antibacterial activity. It serves as a toxic component of P. aeruginosa to ensure P. aeruginosa gets the advantageous position when competing with other bacterial species. Tli5PA is the innate immunity protein of PldA and protects it from the toxicity of PldA. Jiang et al. reported that PldB was the effector of P. aeruginosa H3-T6SS and its three immunity proteins were encoded by PA5088, PA5087, and

Fig. 2. Gene clusters of P. aeruginosa T6SSs. The homologous components between them are marked with the same color. Each gene is marked with its name. dot, defect in organelle trafficking; fha, forkhead-associated protein; hcp, haemolysin coregulated protein; his, hcp secretion island; icm, intracellular multiplication; lip, lipoprotein; ppk, Pseudomonas protein kinase; ppp, Pseudomonas protein phosphatase; sfa, sigma factor activator; stk, Ser/Thr kinase; stp, Ser/Thr phosphatase; vgrG, valine–glycine repeats protein (Filloux et al., 2008).

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Table 1 The genes involved in P. aeruginosa T6SSs. T6SS loci

Components

Characteristics

Refs

HSI-I, II, III HSI-I, II, III HSI-I, II

ClpV DotU (TssL) Fha

Mougous et al. (2006) Boyer et al. (2009) Mougous et al. (2007)

HSI-I, II, III

Hcp

As the energy source facilitating Hcp secretion Encoding homologs of T4SS stabilizing proteins A forkhead-associated domain protein,its phosphorylation can trigger Hcp secretion A hexameric protein, T6SS-dependent secreted protein

HSI-I, II, III HSI-I

HsiA HsiB1C1 (TssB/TssC)

Unknown Similar to VipA/VipB tubules, with a central hole of ∼100 A˚

HSI-I

HsiE1 (TagJ)

HSI-I, II, III HSI-I, II, III HSI-I, II, III HSI-I, II, III

HsiF (TssE) HsiG HsiH HsiJ (TssJ)

HSI-I, II, III HSI-I, II, III HSI-I HSI-II, III

IcmF (TssM) lip PppA/PpkA Sfa

HSI-II HSI-I, II, III

Stp1/Stk1 VgrG

Interact with HsiB1 to form a novel subcomplex of the T6SS A gp25-like protein, but not exhibit lysozyme activity Unknown Unknown A protein possesses a compact ␤-sandwich fold and presents at 1.4 A˚ resolution Encoding homologs of T4SS stabilizing proteins Unknown Regulates hcp1 secretion in transcriptional level The putative enhancer binding proteins encoded on H2-T6SS and H3-T6SS left Encoding an IcmF-like protein Similar to the tail spike, are not only secreted components but are essential for secretion of other T6SS substrates

PA5086 (Jiang et al., 2014). PldB is active in the bacterial periplasm and exerts an antibacterial activity. In addition, PldB can also facilitate intracellular invasion of host eukaryotic cells by activating the phosphatidylinositol 3 kinase (PI3K)/Akt pathway, suggesting that it is a trans-kingdom effector.

Mougous et al. (2006) and Osipiuk et al. (2011) Lossi et al. (2012) and Lossi et al. (2013) Lossi et al. (2012) Lossi et al. (2011)

Robb et al. (2013) Boyer et al. (2009) Mougous et al. (2007) Sana et al. (2013) Mukhopadhyay et al. (1999) Hachani et al. (2011)

strain. Thus H3-T6SS is also required for virulence in C. elegans (Sana et al., 2013). While the mutation of HSI-I could attenuate or abolish the lung infection in a rat model of chronic respiratory infection caused by P. aeruginosa (Potvin et al., 2003). 4.3. Takes part in bacterial biofilm formation

4. Function of P. aeruginosa T6SS 4.1. Delivery of toxin to other competing bacterial species to help the growth of P. aeruginosa P. aeruginosa lives extensively in various environments and has survival advantage when there are multiple bacterial species. The survival advantage of P. aeruginosa is associated with T6SS. In 2010, Hood et al. identified three substrates of P. aeruginosa H1-T6SS, i.e. Tse1-3 (Hood et al., 2010). To protect P. aeruginosa from the damage by Tse2, the cognate immunity of Tsi2 can block the activity of Tse2 through direct or indirect interaction of the proteins. In addition, as a toxin component of the toxin immunity system, Tse2 could arrest the growth of host cells to protect P. aeruginosa when the eukaryotic or prokaryotic cells without Tsi2 were injected with Tse2. Further research has shown that T6SS-dependent antibacterial activity of P. aeruginosa is triggered by the activity of T6SS displayed by predatory bacteria. P. aeruginosa could not efficiently kill T6SS− V. cholerae or Acinetobacter baylyi. However, when V. cholerae or A. baylyi expressed functional T6SS activity, they could be attacked by P. aeruginosa. The “tit-for-tat” evolutionary strategy ensures P. aeruginosa can effectively counterattack heterologous T6SS+ species that coexist in the same ecological niche; on the other hand, P. aeruginosa does no harm to “the peaceful bystanders” although they also come in contact closely (Basler et al., 2013). 4.2. As the virulence factor of P. aeruginosa In one Caenorhabditis elegans survival experiment, the survival percentage of C. elegans infected with PAO1 was lower than that of clpV2 mutant, suggesting that H2-T6SS contributes to the virulence of P. aeruginosa (Sana et al., 2012). The other experiment showed that C. elegans infected with PAO1 clpV3 mutant appeared to die with a 2 days delay when compared to the wild-type (WT)

Southey-Pillig et al. first examined the expression of Hcp in P. aeruginosa PAO1 biofilm maturation stage through twodimensional gel electrophoresis and mass spectrometry, which confirmed that Hcp took part in biofilm formation in P. aeruginosa (Southey-Pillig et al., 2005). In addition, in P. aeruginosa PA14 WT strain, the expression of Hcp1 in biofilm cells was higher than that of planktonic cells. When Hcp1 was deleted, biofilm-specific antibiotic resistance was attenuated, suggesting that H1-T6SS was associated with biofilm-specific antibiotic resistance (Zhang et al., 2011). 5. Regulation of P. aeruginosa T6SS 5.1. Transcriptional regulation The expression of P. aeruginosa T6SS at the transcriptional level is regulated by quorum sensing (QS) regulator LasR. QS differentially regulated three T6SSs of P. aeruginosa. The gene expression of HSI-I is suppressed by both the 4-hydroxy-2-alkylquinoline transcriptional regulator MvfR and the homoserine lactone transcription factor LasR. On the contrary, MvfR and LasR can positively regulate the gene expression of both HSI-II and HSI-III. However, there are no LasR binding sites in either HSI-I or HSI-III, suggesting that LasR regulates these loci indirectly (Lesic et al., 2009). Sana et al. also reported that the gene expression of H2-T6SS P. aeruginosa PAO1 WT strain is upregulated by the Las and Rhl QS systems (Sana et al., 2012). 5.2. Posttranscriptional regulation Posttranscriptional regulation of P. aeruginosa T6SS is executed by RNA binding protein RsmA. Brencic and Lory used genomic microarray analysis to compare the altered gene expression in

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Fig. 3. Model of P. aeruginosa H1-T6SS posttranslational regulatory pathways. Donor (blue), P. aeruginosa; recipient (brown), a competing Gram-negative bacterium; IM, inner membrane; OM, outer membrane; P, periplasm. (Silverman et al., 2011). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

P. aeruginosa PAK RsmA mutant strain with P. aeruginosa PAK WT strain, and found that the gene expression of ppkA, ppA, fhA (forkhead-associated), hcp1, and clpV1 was upregulated (Brencic and Lory, 2009). They further used gel shift analyses to identify that HSI-I of P. aeruginosa was directly negatively regulated by RsmA and the hybrid sensor kinase RetS was part of RsmA regulation pathway. RetS is a multifunctional regulator that regulates multiple virulence phenotypes associated with P. aeruginosa acute infection and chronic persistence such as the activation of T3SS and the formation of biofilm. RetS-dependent regulation was implicated in the GacS/GacA/rsmZ signal transduction pathway (Goodman et al., 2004). Conversely, the other hybrid sensor LadS mediates the opposite regulation in the GacS/GacA/rsmZ pathway compared with RetS, such as the suppression of secretion of T3SS and the promotion of biofilm formation. Both RetS and LadS execute their functions by influencing the level of small RNA-RsmZ, an antagonist of RsmA (Ventre et al., 2006). GacS/GacA is a type of two-component system. It is a key mediator in the adaptation of microorganisms to the environments and can regulate the expression of a variety of phenotypes such as the production of extracellular enzymes, QS molecules, secreted toxins, various metabolic functions, motility, etc. GacS/GacA system controls the expression of target genes by regulating the expression of two downstream small RNA, i.e. rsmY and rsmZ (Brencic et al., 2009). Further research demonstrated that gets mutation not only induces formation of biofilm, but also displays high levels of cyclic-di-GMP (c-di-GMP), a second messenger. c-di-GMP is the sensor of the bacterial transition from planktonic to sessile. High levels of c-di-GMP promote the formation of biofilm and chronic infection. RetS mutant leads to repression of the T3SS but upregulation of the T6SS. When the level of c-di-GMP was artificially changed, the production of T3SS and T6SS could be switched,

and RsmY and RsmZ were required for the c-di-GMP-dependent T3SS/T6SS switch, suggesting that there was a firm link between the RetS/GacS and c-di-GMP pathways (Moscoso et al., 2011). 5.3. Posttranslational regulation Posttranslational regulation of P. aeruginosa T6SS includes threonine phosphorylation (TPP)-dependent and TPP-independent pathways. After the discovery that there are three loci in P. aeruginosa by the research team of Mougous in 2006, the same research team showed that the phosphorylation of PpkA, a serinethreonine (Ser-Thr) kinase, can regulate the secretion of Hcp1 required for the assembly of the P. aeruginosa H1-T6SS. PppA, a Ser-Thr phosphatase, can antagonize PpkA. The reciprocal effects on the P. aeruginosa H1-T6SS was through its action on Fha1, an FHA domain-containing protein. In brief, environmental cue activates, dimerizes, and autophosphoryates PpkA, a membrane-spanning threonine kinase. The autophosphorylation of PpkA causes binding and subsequent phosphorylation of Fha1, which is required for the secretion of Hcp1 (Mougous et al., 2007). This route was identified as a TPP-dependent pathway for the posttranslational regulation of P. aeruginosa H1-T6SS, even though the environment cue to induce PpkA dimerization is still unknown. In 2009, the same research team discovered that TagR (type VI secretion associated gene R; PA0071) is the upstream component of P. aeruginosa T6SS posttranslational regulatory pathway and acts on PpkA to promote its activity (Hsu et al., 2009). Furthermore in 2011, they found that as a posttranslational regulation protein, TagF can repress the activation of P. aeruginosa H1-T6SS (Silverman et al., 2011). Interestingly, TagF regulates the activity of the P. aeruginosa H1-T6SS in a manner significantly different from previous TPP pathway, which is called

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TPP-independent pathway. These two posttranslational regulation pathways are depicted in Fig. 3. 5.4. Iron Many pathogens live in an iron-deficient host environment, where a lot of gene transcriptions are regulated by iron through the ferric-uptake regulator (Fur) protein. Fur is a key modulator of iron-dependent gene expression in bacteria. In P. aeruginosa, Sana et al. determined that there were two putative Fur boxes in the promoter of H2-T6SS, and the transcription of H2-T6SS was repressed by iron (Sana et al., 2012). 5.5. 54 factor (RpoN) P. aeruginosa have three T6SS loci and each T6SS is regulated by divergent mechanism. Sana et al. reported that at the transition between the exponential and stationary phases, both the expressions of H2-T6SS and H3-T6SS are induced by QS, while H3-T6SS reached full expression later than H2-T6SS in the stationary phase. In addition, the two T6SSs are controlled by RpoN (␴54 factor). H2-T6SS and the right H3-T6SS operon are negatively regulated by RpoN, while the left H3-T6SS operon is positively regulated by RpoN. The gene encoded RpoN activator is called Sfa (sigma factor activator). The H2-T6SS operon is indirectly controlled by RpoN in an Sfa2-dependent manner, but the expression of H3-T6SS is Sfa independent and may be regulated by RpoN/GacA/RsmA pathway (Sana et al., 2013). 6. Concluding remarks In recent years, the research on P. aeruginosa T6SS has attracted much attention and has become a hot field in microbiology. Rapid progress has been made in the structure and biogenesis of T6SS in P. aeruginosa, but there are still many questions to be elucidated. For example, what are the molecular mechanisms when P. aeruginosa T6SS recognizes the target cell? As P. aeruginosa T6SS secretes multiple effectors, are they all secreted at the same time or are there any selective regulation mechanisms? Are there any new effectors to be found? Can the P. aeruginosa T6SS be beneficial to its neighboring bacteria but not just be competitive? In addition, P. aeruginosa T6SS can secrete toxic proteins into eukaryotic cells, which is absent in other secretion systems, suggesting its functional diversity. The critical role of this system in bacterial pathogenesis and bacteria-host interation gives an encouraging outlook for us. For example, in the age of synthetic biology could this T6SS be used for attacking of other cells to suppress horizontal genetic elements transfer in bacterial populations. It also may be the potential target of novel antimicrobials to fight the human polymicrobial infections. With further research, the progress of P. aeruginosa T6SS will no doubt bring us surprise and new challenges, and also provide important influence in ecology, agriculture, and medicine. References Basler M, Ho BT, Mekalanos JJ. Tit-for-tat. Type VI secretion system counterattack during bacterial cell–cell interactions. Cell 2013;152(4):884–94. Benz J, Sendlmeier C, Barends TR, Meinhart A. Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa. PLoS ONE 2012;7(7):e40453, http://dx.doi.org/10.1371/journal.pone.0040453. Bingle LEH, Bailey CE, Pallen MJ. Type VI secretion: a beginner’s guide. Curr Opin Microbiol 2008;11(1):3–8. Bleves S, Viarre V, Salacha R, Michel GP, Filloux A, Voulhoux R. Protein secretion systems in Pseudomonas aeruginosa: a wealth of pathogenic weapons. Int J Med Microbiol 2010;300(8):534–43. Bönemann G, Pietrosiuk A, Mogk A. Tubules and donuts. A type VI secretion story. Mol Microbiol 2010;76(4):815–21.

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