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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Host Microbe. Author manuscript; available in PMC Jan 21, 2011.
Published in final edited form as:
PMCID: PMC2831478
NIHMSID: NIHMS168191

A Type VI Secretion System of Pseudomonas aeruginosa Targets a Toxin to Bacteria

Abstract

The functional spectrum of a secretion system is defined by its substrates. Here we analyzed the secretomes of Pseudomonas aeruginosa mutants altered in regulation of the Hcp Secretion Island-I-encoded type VI secretion system (H1-T6SS). We identified three substrates of this system, proteins Tse1-3 (type six exported 1-3), which are coregulated with the secretory apparatus and secreted under tight posttranslational control. The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 had no effect on eukaryotic cells; however, it provided a major growth advantage for P. aeruginosa strains, relative to those lacking immunity, in a manner dependent on cell contact and the H1-T6SS. This demonstration that the T6SS targets a toxin to bacteria helps reconcile the structural and evolutionary relationship between the T6SS and the bacteriophage tail and spike.

INTRODUCTION

Secreted proteins allow bacteria to intimately interface with their surroundings and other bacteria. The importance and diversity of secreted proteins is reflected in the multitude of pathways bacteria have evolved to enable their export (Abdallah et al., 2007; Cascales, 2008; DiGiuseppe Champion and Cox, 2007; Filloux et al., 2008; Thanassi and Hultgren, 2000). Large multi-component secretion systems, including types III and IV secretion, have been the focus of a great deal of study because in many organisms they are specialized for effector export and they have the remarkable ability to directly translocate proteins from bacterial to host cell cytoplasm via a needle-like apparatus (Cambronne and Roy, 2006; Cascales and Christie, 2003; Galan, 2009). The recently described type VI secretion system (T6SS) is another specialized system, however its physiological role and general mechanism remain poorly understood (Bingle et al., 2008).

Studies of T6SSs indicate that a functional apparatus requires the products of approximately 15 conserved and closely linked genes, and is strongly correlated to the export of a hexameric ring-shaped protein belonging to the hemolysin co-regulated protein (Hcp) family (Aschtgen et al., 2008; Mougous et al., 2006; Mougous et al., 2007; Pukatzki et al., 2006; Zheng and Leung, 2007). Hcp proteins are required for assembly of the secretion apparatus and they interact with valine-glycine repeat (Vgr) family proteins, which are also exported by the T6SS. The function of the Hcp/Vgr complex remains unclear, however it is believed that the proteins are extracellular structural components of the secretion apparatus. Recent X-ray crystallographic insights into Hcp and Vgr-family proteins show that they are similar to bacteriophage tube and tail spike proteins, respectively (Leiman et al., 2009; Mougous et al., 2006; Pell et al., 2009). These findings have prompted speculation that the T6SS is evolutionarily, structurally, and mechanistically related to bacteriophage. According to this model, the T6SS assembles as an inverted phage tail on the surface of the bacterium, with the Hcp/Vgr complex forming the distal end of the cell-puncturing device (Kanamaru, 2009). Another notable conserved T6S gene product is ClpV, a AAA+-family ATPase that has been postulated to provide the energy necessary to drive the secretory apparatus (Bonemann et al., 2009; Mougous et al., 2006). The roles of the remaining conserved T6S proteins remain largely unknown.

Nonconserved genes encoding predicted accessory elements are also linked to most T6SSs (Bingle et al., 2008; Mougous et al., 2007; Shalom et al., 2007). In the case of the HSI-I-encoded T6SS of Pseudomonas aeruginosa (H1-T6SS) (Figure 1A), the subject of this report, these genes encode elements of a posttranslational regulatory pathway. These proteins strictly modulate the activity of the secretion system through changes in the phosphorylation state of a forkhead-associated domain protein, Fha1 (Mougous et al., 2007). Phosphorylation of Fha1 by a transmembrane serine-threonine Hanks-type kinase, PpkA, triggers Hcp1 secretion. PppA, a PP2C-type phosphatase, antagonizes Fha1 phosphorylation.

Figure 1
Overview and results of an MS-based screen to identify H1-T6SS substrates

The T6SS has been linked to a myriad of processes, including biofilm formation (Aschtgen et al., 2008; Enos-Berlage et al., 2005), conjugation (Das et al., 2002), quorum sensing regulation (Weber et al., 2009), and both promoting and limiting virulence (Cascales, 2008; Filloux, 2009; Pukatzki et al., 2009; Yahr, 2006). The P. aeruginosa H1-T6SS has been implicated in the fitness of the bacterium in a chronic infection; mutants in conserved genes in this secretion system failed to efficiently replicate in a rat lung chronic infection model and the system was shown to be active in cystic fibrosis (CF) patient infections (Mougous et al., 2006; Potvin et al., 2003). The H1-T6SS is also co-regulated with other chronic infection virulence factors such as the psl and pel loci, which are involved in biofilm formation (Goodman et al., 2004; Ryder et al., 2007).

How the apparently conserved T6SS architecture can participate in such a wide range of activities is not clear. At least one mechanism by which the secretion system can exert its effects on a host cell has been garnered from studies of Vibrio cholerae. A T6S-associated VgrG-family protein of this organism contains a domain with actin-crosslinking activity that is translocated into host cell cytoplasm in a process requiring endocytosis and cell-cell contact (Ma et al., 2009; Pukatzki et al., 2007; Satchell, 2009). The subset of VgrG-family proteins that contain non-structural domains with conceivable roles in pathogenesis have been termed “evolved” VgrG proteins (Pukatzki et al., 2007). This configuration, wherein an effector domain is presumably translocated into host cell cytoplasm by virtue of its fusion to the T6S cell puncturing apparatus, is intriguing, but it is likely not general; a multitude of organisms containing T6SSs do not encode “evolved” VgrG proteins (Boyer et al., 2009; Pukatzki et al., 2009).

Key to understanding the function of the T6SS – as with any secretion system – is to identify and characterize the protein substrates that it exports. EvpP from Edwardsiella tarda and RbsB from Rhizobium leguminosarum are the only proposed substrates of the system to date; however, whether these proteins are true substrates remains an open question. Inconsistent with anticipated properties of T6S substrates, RbsB contains an N-terminal Sec secretion signal, and EvpP stably associates with a component of the secretion apparatus (Bladergroen et al., 2003; Pukatzki et al., 2009; Zheng and Leung, 2007).

In this study, we identified three proteins, termed Tse1-3 (type VI secretion exported 1-3), that are substrates of the H1-T6SS of P. aeruginosa. We showed that one of these, Tse2, is the toxin component of a toxin-immunity system, and that it is able to arrest the growth of a variety of prokaryotic and eukaryotic organisms. Despite the promiscuity of toxin expressed intracellularly, we found that H1-T6SS-exported Tse2 was specifically targeted to bacteria. In growth competition experiments, immunity to Tse2 provided a marked growth advantage in a manner dependent on intimate cell-cell contact and a functional H1-T6SS. The ability of the secretion system to efficiently target Tse2 to a bacterium, and not to a eukaryotic cell, suggests that T6S may play a role in the delivery of toxin and effector molecules between bacteria.

RESULTS

Design and Characterization of H1-T6SS On- and Off-State Strains

Under laboratory culturing conditions, activation of the H1-T6SS is strongly repressed at the posttranslational level by the phosphatase PppA (Figure 1A). We have shown that inactivation of pppA leads to Hcp1 export, and that this could reflect triggering of the “on-state” in the secretory apparatus (Hsu et al., 2009; Mougous et al., 2007). These observations led us to predict that additional components of the apparatus, and even substrates of the secretion system, are also exported in this state. To identify these proteins, we sought to compare the secretomes of ΔpppA and ΔclpV1. The latter lacks the H1-T6SS ATPase, ClpV1, and therefore remains in the “off-state” (Figure 1A) (Mougous et al., 2006).

To probe whether the on-state and off-state mutations could modulate the activity of the H1-T6SS, we assayed their effect on Hcp1 secretion in P. aeruginosa PAO1 hcp1V (where present, –V denotes a fusion of the indicated gene to a sequence encoding the vesicular stomatitis virus G epitope). As expected, the deletion of pppA promoted Hcp1 secretion and Fha1 phosphorylation relative to the parental strain (Figure 1B and C). Since the wild-type strain does not secrete Hcp1 to detectable levels, the effects of ΔclpV1 were gauged using the ΔpppA background. Introduction of the clpV1 deletion to ΔpppA abrogated Hcp1 secretion and this effect was fully complemented by ectopic expression of clpV1 (Figure 1B). These data indicate that pppA and clpV1 deletions are sufficient to activate and inactivate the H1-T6SS secretion system, respectively.

Mass Spectrometric Analysis of On- and Off-State Secretomes

Next, we used MS and spectral counting to compare proteins present in the secretomes of the on- and off-state P. aeruginosa strains (Liu et al., 2004). Average spectral count (SC) values were used to identify whether each protein was differentially secreted between states. The results of our MS analyses are summarized in Table S1. Importantly, the total number of spectral counts was comparable between the on- and off-states in both replicates. A total of 371 proteins that met our filtering criteria were identified between replicate experiments (Tables S2). We divided the proteins into three groups: Category 1 (C1; Tables S3 and S4) – present in both the on- and off-states, Category 2 (C2; Table S5) – present only in the on-state, and Category 3 (C3; Table S6) – present only in the off-state. Overlap between the replicates was greatest among C1 proteins. A total of 314 C1 proteins were identified, of which 249 were shared between the replicates. A significant fraction of the C1 differences can be ascribed to the fact that 13% more proteins were identified in this category in Replicate 1 (R1) than in Replicate 2 (R2).

To assess the accuracy of the quantitative component of our datasets, we measured the distribution of SC ratios (on-state/off-state) within C1 proteins (Figure 1D). Since we did not anticipate that the H1-T6SS should exhibit a global effect on the secretome, we were encouraged by the approximate split (50% ± 2 in both replicates) between those proteins that were up- versus down-regulated between the on- and off-states. Additionally, the change in average SCs between the states was low, and this value was similar in the replicates ([R1], 1.13 ± 1.04; [R2], 1.15 ± 0.90). Only 30 R1 and 33 R2 proteins yielded a SC ratio > 2.

As expected, Hcp1 was over-represented in the on-state samples. Indeed, Hcp1 was the most differentially secreted protein in both datasets (SC ratio: [R1], 13; [R2], 17]) (Figure 1D). The presence of Hcp1 in the secretome of off-state cells suggests a certain extent of cellular protein contamination within the preparations. This contamination is also evidenced by the predicted or known functions of many of the detected proteins (Tables S2–S4). The high abundance of Hcp1 (119 SC average) relative to the average protein abundance (10.9 SC) is likely another factor contributing to its detection in the off-state samples.

Next we analyzed C2 proteins – those observed only in the on-state. Similar numbers of these proteins were identified in R1 (19) and R2 (20), and five of these were found in both replicates (Table 1). The reproducibility of C2 versus C1 proteins is attributable to the difference in their average SCs; the average SC of C2 proteins was 2.6, versus 12 in C1. The C2 proteins identified in both R1 and R2 accounted for five of the six most abundant in C2–R1, and five of the ten most abundant in C2–R2. Each of these proteins lacked a secretion signal for known export pathways. The identity of these proteins and the biochemical validation of their secretion is the subject of subsequent sections.

Table 1
Select parameters of Category 2 and 3 proteins detected in both replicates of MS secretome analyses.

The number and abundance of C3 proteins in both R1 and R2 was slightly lower than the corresponding C2 values. Nonetheless, we did identify three C3 proteins in common between R1 and R2 (Table 1). The occurrence of these proteins in the off-state is likely to reflect changes in gene regulation caused by modulation of the activity of the H1-T6SS that manifest in the secretome. Sequence analysis indicated that each of these proteins contains a predicted signal peptide (Emanuelsson et al., 2007).

Two VgrG Proteins are Secreted by the H1-T6SS

Two VgrG-family proteins, the products of open reading frames PA0091 and PA2685, were the most abundant C2 proteins in R1 and R2 (Table 1). Interestingly, earlier microarray work has shown that PA0091 and PA2685 are coordinately regulated with HSI-I by the RetS hybrid two-component sensor/response regulator protein, however the participation of these proteins in the H1-T6SS was not investigated (Figure 2A) (Goodman et al., 2004; Laskowski and Kazmierczak, 2006; Zolfaghar et al., 2005). The PA0091 locus is located within HSI-I, while the PA2685 locus is found at an unlinked site that lacks other apparent T6S elements (Figure 1A and and2A).2A). To remain consistent with previous nomenclature, these genes will henceforth be referred to as vgrG1 and vgrG4 (Mougous et al., 2006).

Figure 2
Two VgrG-family proteins are regulated by retS and secreted in an H1-T6SS-dependent manner

To confirm the MS results, we compared the localization of VgrG1 and VgrG4 in wild-type bacteria to strains containing the on-state (ΔpppA) and off-state (ΔclpV1) mutations. Consistent with our MS findings, Western blot analyses of cell and supernatant fractions in vgrG1V and vgrG4V backgrounds indicated that secretion of the proteins is strongly repressed by pppA and requires clpV1 (Figure 2B and 2C). These data show that the H1-T6SS exports at least two VgrG-family proteins. For reasons not yet understood, VgrG4–V migrated as two major bands in the cellular fraction and a large number of high molecular weight bands in the supernatant.

Identification of Three H1-T6SS Substrates

The remaining C2 proteins identified in both R1 and R2 are hypothetical proteins encoded by ORFs PA1844, PA2702, and PA3484. Bioinformatic analyses of these proteins indicated that they do not share detectable sequence homology to each other or to proteins outside of P. aeruginosa. Each protein is encoded by an ORF that resides in a predicted two-gene operon with a second hypothetical ORF. Intriguingly, we noted that the three unlinked operons – like HSI-I (which includes vgrG1) and vgrG4 – are negatively regulated by RetS (Figure 2A).

Based on our secretome analyses, we hypothesized that the proteins encoded by PA1844, PA2702, and PA3484, henceforth referred to Tse1-3, respectively, are substrates of the H1-T6SS. To test this, we analyzed the localization of the proteins when ectopically expressed in a diagnostic panel of P. aeruginosa strains. The secretion profile of each protein was similar in these strains; relative to the wild-type, ΔpppA displayed dramatically increased levels of secretion, and secretion levels were at or below wild-type levels in ΔpppA strains containing additional deletions in either hcp1 or clpV1 (Figure 3A). Over-expression of the proteins was ruled out as a confounding factor, as the secretion profile of chromosomally-encoded Tse1–V in related backgrounds was similar to that of the ectopically-expressed protein (Figure 3B). Finally, we complemented Tse1–V secretion in ΔpppA ΔclpV1 tse1V with a plasmid expressing clpV1.

Figure 3
The Tse proteins are tightly regulated H1-T6SS substrates

To further distinguish the Tse proteins as H1-T6SS substrates rather than structural components, we determined their influence on core functions of the T6 secretion apparatus. Fundamental to each studied T6SS is the ability to secrete an Hcp-related protein (Cascales, 2008). In a systematic analysis, Hcp secretion was shown to require all predicted core T6SS components, including VgrG-family proteins (Pukatzki et al., 2007; Zheng and Leung, 2007). We generated a strain containing a deletion of all tse genes in the ΔpppA hcp1V background and compared Hcp1 secretion in this strain to strains lacking both vgrG1 and vgrG4 or clpV1 in the same background. Western blot analysis revealed that Hcp1 secretion was abolished in both the ΔclpV1 and ΔvgrG1 ΔvgrG4 strains, however it was unaffected by tse deletion (Figure 3C).

A multiprotein complex containing ClpV1 is essential for a functional T6S apparatus (Hsu et al., 2009). As a second indicator of H1-T6SS function, we used fluorescence microscopy to examine the formation of this complex in strains containing a chromosomal fusion of clpV1 to a sequence encoding the green fluorescent protein (clpV1GFP) (Mougous et al., 2006). In line with the Hcp1 secretion result, the punctate appearance of ClpV1–GFP localization, which is indicative of proper apparatus assembly, was not dependent on the tse genes (Figure 3D). On the other hand, deletion of ppkA, a gene required for assembly of the H1-T6S apparatus, disrupted ClpV1–GFP localization. Together, these findings provide evidence that the Tse proteins are substrates of H1-T6SS.

Tse Secretion is Triggered by De-Repression of the Gac/Rsm Pathway

Earlier microarray experiments suggested that the tse genes are tightly repressed by RetS, a component of the Gac/Rsm signaling pathway (Lapouge et al., 2008). In this pathway, the activity of RetS and two other sensor kinase enzymes, LadS and GacS, converge to reciprocally regulate an overlapping group of acute and chronic virulence pathways in P. aeruginosa through the small RNA-binding protein RsmA (Brencic and Lory, 2009; Brencic et al., 2009; Goodman et al., 2004; Ventre et al., 2006; Yahr and Greenberg, 2004). To directly investigate the effect of the Gac/Rsm pathway on tse expression, we monitored the abundance of Tse proteins in the cell-associated and secreted fractions of strains containing the retS deletion. Our data showed that activation of the Gac/Rsm pathway dramatically elevates cellular Tse levels and triggers their export via the H1-T6SS (Figure 3E). It is noteworthy that secretion of Tse proteins in ΔretS is far in excess of that observed in ΔpppA (Figure 3E, compare ΔpppA and ΔretS).

Tsi2 is an Essential Protein that Protects P. aeruginosa from Tse2

The lack of transposon insertions within the tse2/tsi2 locus in a published transposon insertion library of P. aeruginosa PAO1 suggested that these ORFs may be essential for viability of the organism (Jacobs et al., 2003). To test this possibility, we attempted to generate deletions of tse2 and tsi2. While a Δtse2 strain was readily constructed, tsi2 was refractory to several methods of deletion. Based on genetic context and co-regulation (Figure 2A), we hypothesized that Tse2 and Tsi2 could interact functionally, and that the requirement for tsi2 could therefore depend on tse2. Success in simultaneous deletion of both genes confirmed this hypothesis (Figure 4A).

Figure 4
The Tse2 and Tsi2 proteins are a toxin-immunity module

Our findings implied that Tsi2 protects cells from Tse2. To probe this possibility further, we introduced tse2 to the Δtse2 Δtsi2 background. Induction of tse2 expression completely abrogated growth of Δtse2 Δtsi2, however it had only a mild effect on wild-type cells. These data demonstrate that tse2 encodes a toxic protein capable of inhibiting the growth of P. aeruginosa, and that tsi2 encodes a cognate immunity protein. We named Tsi2 based on this property (type VI secretion immunity protein 2).

Tsi2 could block the activity of Tse2 through a mechanism involving direct interaction of the proteins, or by an indirect mechanism wherein the proteins function antagonistically on a common pathway. To determine if Tse2 and Tsi2 physically interact, we conducted co-immunoprecipitation studies in P. aeruginosa. Tse2 was specifically identified in precipitate of Tsi2–V, indicative of a stable Tse2-Tsi2 complex (Figure 4B). These data provide additional support for a functional interaction between Tse2 and Tsi2, and they suggest that the mechanism of Tsi2 inhibition of Tse2 is likely to involve physical association of the proteins.

Intracellular Tse2 is Toxic to a Broad Spectrum of Prokaryotic and Eukaryotic Cells

P. aeruginosa is widely dispersed in terrestrial and aquatic environments, and it is also an opportunistic pathogen with a diverse host range. As such, Tse2 exported from P. aeruginosa has the potential to interact with a range of organisms, including prokaryotes and eukaryotes. To investigate the organisms that Tse2 might target, we expressed tse2 in the cytoplasm of representative species from each domain. Two eukaryotic cells were chosen for our investigation, Saccharomyces cerevisiae and the HeLa human epithelial-derived cell line. Yeast were included primarily for diversity, however these organisms also interact with P. aeruginosa in assorted environments and could therefore represent a target of the toxin (Wargo and Hogan, 2006). S. cerevisiae cells were transformed with a galactose-inducible expression plasmid for each tse gene, or with an empty control plasmid (Mumberg et al., 1995). Relative to the other tse genes and the control, tse2 expression caused a dramatic decrease in observable colony forming units following 48 hrs of growth under inducing conditions (Figure 5A). To address the specificity of Tse2 effects on S. cerevisiae, we next tested whether Tsi2 could block Tse2-mediated toxicity. Co-expresssion of tsi2 with tse2 restored viability to levels similar to the control strain (Figure 5B). This result implies that the effects of Tse2 on S. cerevisiae are specific and that the toxin may act via a similar mechanism in bacteria and yeast. Our findings are consistent with an earlier screen for P. aeruginosa proteins toxic to yeast. Arnoldo et al. found Tse2 among nine P. aeruginosa proteins most toxic to S. cerevisiae within a library of 505 that included known virulence factors (Arnoldo et al., 2008).

Figure 5
Heterologously expressed Tse2 is toxic to prokaryotic and eukaryotic cells

The effects of Tse2 on a mammalian cell were probed using a reporter co-transfection assay in HeLa cells. Expression plasmids containing the tse genes were generated and mixed with a GFP reporter plasmid. Co-transfection of the reporter plasmid with tse1 and tse3 had no impact on GFP expression relative to the control; however, inclusion of the tse2 plasmid reduced GFP expression to background levels (Figure 5C and 5D). We also noted morphological differences between cells transfected with tse2 and control transfections, which was apparent in the fraction of rounded cells (Figure 5E). These were specific effects of Tse2, as the inclusion of a tsi2 expression plasmid into the tse2/GFP reporter plasmid transfection restored GFP expression and lowered the fraction of rounded cells to the control. From these studies, we conclude that Tse2 has a deleterious effect on essential cellular processes in assorted eukaryotic cell types.

Next we asked whether Tse2 has activity in prokaryotes other than P. aeruginosa. We tested two organisms, Escherichia coli and Burkholderia thailandensis. Both organisms were transformed with plasmids engineered for inducible expression of either tse2, or as a control, both tse2 and tsi2. In each case, tse2 expression strongly inhibited growth and co-expression with tsi2 reversed this effect (Figures 5F and 5G). Taken together with the effects we observed in S. cerevisiae and HeLa cells, we conclude that Tse2 is a toxin that – when administered intracellularly – inhibits essential cellular processes in a broad spectrum of organisms.

P. aeruginosa can target bacterial, but not eukaryotic cells, with Tse2

Mounting evidence indicates that the T6SS is capable of cell contact-dependent protein translocation into the cytoplasm of a eukaryotic cell (Ma et al., 2009; Pukatzki et al., 2007; Suarez et al., 2009). Since tse2 expression experiments indicated that the toxin could act on eukaryotes (Figure 5A–E), we asked whether P. aeruginosa could target these cells with the H1-T6SS. We measured cytotoxicity toward HeLa cells for a panel of P. aeruginosa strains, including Tse2 hyper-secreting (ΔretS) and non-secreting backgrounds (ΔretS ΔclpV1). Under all conditions analyzed, including the use of centrifugation to encourage contact between the bacterial and mammalian cells, we were unable to observe Tse2-promoted cytotoxicity or a morphological impact on the cells as was observed in transfection experiments (Figure 6A). Similar negative results were obtained using the J774 mouse macrophage cell line. Additionally, attempts to detect Tse2 or other Tse proteins in host cell cytoplasm using differential lysis strategies or host protein-dependent phosphorylation tags yielded no evidence of H1-T6SS-dependent protein targeting to mammalian cells (data not shown). We also investigated Tse2-dependent effects on yeast co-cultured with P. aeruginosa. Under all conditions analyzed, including those designed to favor intimate cell contact, no effect could be attributed to Tse2 (Figure S1). Based on our data, we concluded that P. aeruginosa is unlikely to utilize Tse2 as a toxin against eukaryotic cells. This conclusion is in-line with the results of several earlier reports, which have shown that P. aeruginosa strains lacking retS are highly attenuated in an assortment of acute virulence-related phenotypes, including macrophage and epithelial cell cytotoxicity (Goodman et al., 2004; Zolfaghar et al., 2005), and acute pneumonia (Laskowski et al., 2004) and corneal infections in mice (Zolfaghar et al., 2006).

Figure 6
Immunity to Tse2 provides a growth advantage against P. aeruginosa strains secreting the toxin by the H1-T6SS

The strong influence of intracellular tse2 expression on the growth of assorted bacteria prompted us to next investigate whether its target could be another prokaryotic cell. To test this, we conducted a series of in vitro growth competition experiments with P. aeruginosa strains in the ΔretS background engineered with regard to their ability to produce, secrete, or resist Tse2. Competitions between these strains were conducted for five hours at 37 °C in liquid medium or following filtration onto a porous solid support. Neither production nor secretion of Tse2, nor immunity to the toxin, impacted the growth rates of competing strains in liquid medium experiments (Figure 6B). On the contrary, a striking proliferative advantage dependent on tse2 and tsi2 was observed when cells were grown on a solid support. In growth competition experiments between ΔretS and ΔretS Δtse2 Δtsi2, henceforth referred to as donor and recipients strains, respectively, donor cells were approximately 14-fold more abundant at the conclusion of the 5 hr period (Figure 6B). This was entirely Tse2 mediated, as a deletion of tse2 from the donor strain, or the addition of tsi2 to the recipient strain, abrogated the growth advantage of the donor. We also tested whether the advantage of the donor required Tse2 secretion. Inactivation of clpV1 within the donor strain confirmed that the Tse2-mediated growth advantage requires a functional H1-T6SS (Figure 6B). It is important to note that in each growth competition experiment, the total proliferation of the donor strain remained constant, indicating that Tse2 suppresses growth of the recipient strain.

In order to examine the extent to which Tse2 could facilitate a growth advantage, we conducted long-term competitions between P. aeruginosa strains with and without Tse2 immunity. The competition experiments were initiated with a donor-to-recipient cell ratio of approximately 10:1, which raises the probability that each recipient cell will contact a donor cell. Over the course of 48 hours, the Tse2 donor strain displayed a remarkable 104-fold growth advantage relative to a recipient strain lacking immunity (Figure 6C). These data conclusively demonstrate that the P. aeruginosa H1-T6SS can be used to target Tse2 to another bacterial cell. The differences we observed between competitions conducted in liquid medium versus on a solid support suggest that intimate donor-recipient cell contact is required for Tse2-mediated effects. While we have not directly demonstrated that Tse2 is translocated into recipient cell cytoplasm, it is a likely explanation for our data given that cell contact is required and Tsi2 is a cytoplasmic immunity protein that physically interacts with the toxin (Figure 4B).

DISCUSSION

The T6SS has been implicated in numerous, apparently disparate processes. With few exceptions, the mode-of-action of the secretion system in these processes is not known. Since the T6SS architecture appears highly conserved, we based our study on the supposition that the diverse activities of T6SSs, including T6SSs within a single organism, must be attributable to a diverse array of substrate proteins exported in a specific manner by each system. Our findings support this model; we identified three T6S substrates that lack orthologs outside of P. aeruginosa, and that specifically require the H1-T6SS for their export (Figure 1 and and33).

Bacterial genomes encode a large and diverse array of toxin-immunity protein (TI) systems (Gerdes et al., 2005; Riley and Wertz, 2002). These can be important for plasmid maintenance, stress response, programmed cell death, cell-fate commitment, and defense against other bacteria. Many TI toxins exert their effects via nuclease activity, through membrane depolarization, or by inhibiting DNA replication machinery (Gerdes et al., 2005; Smarda and Smajs, 1998). These activities frequently result in promiscuity against eukaryotic cells, as we observed with Tse2 (Figure 5). However, our findings suggest that the Tse system differs from other TI systems in certain respects. To our knowledge, Tse2 is the only example of a TI system toxin exported through a large, specialized secretion apparatus. Many TI system toxins are either not actively secreted, or they utilize the sec pathway (Riley and Wertz, 2002). This distinction implies that secretion through the T6S apparatus is required to target Tse2 to a relevant environment, cell, or subcellular compartment. Indeed, we have shown that targeting of Tse2 by the T6S apparatus is essential for its activity (Figure 6).

We found that Tse2 is active against assorted bacteria and eukaryotic cells when expressed intracellularly (Figures 4 and and5).5). Despite this, we found no evidence that P. aeruginosa can target Tse2 to a eukaryotic cell, including mammalian cells of epithelial and macrophage origin (Figure 6A and data not shown). Surprisingly, P. aeruginosa efficiently targeted the toxin to another bacterial cell (Figure 6). These findings, combined with the following recent observations, provide support for the hypothesis that the T6SS can serve as an inter-bacterial interaction pathway. First, the secretion system is present and conserved in many non-pathogenic, solitary bacteria (Bingle et al., 2008; Boyer et al., 2009). Second, there is experimental evidence supporting an evolutionary relationship between extracellular components of the secretion apparatus and the tail proteins of bacteriophages T4 and λ (Ballister et al., 2008; Leiman et al., 2009; Pell et al., 2009; Pukatzki et al., 2007). Finally, two recent reports have implicated the conserved T6S component, VgrG, in inter-bacterial interactions. A bioinformatic analysis of Salmonella genomes identified a group of “evolved” VgrG proteins bearing C-terminal effector domains highly related to bacteria-targeting S-type pyocins, and a VgrG protein from Proteus mirabilis was shown to participate in an intra-species self/non-self recognition pathway (Blondel et al., 2009; Gibbs et al., 2008).

It is also evident that in certain instances the T6SS has evolved to engage eukaryotic cells. In at least two reports, the T6S apparatus has been demonstrated to deliver a protein to a eukaryotic cell (Ma et al., 2009; Suarez et al., 2009). Moreover, the T6SSs of several pathogenic bacteria are major virulence factors (Bingle et al., 2008). Taken together with our findings, we posit that there are two broad groups of T6SSs, those that target bacteria and those that target eukaryotes. It is not possible at this time to rule out that a given T6SS may have dual specificity. However, our inability to detect the effects of Tse2 in an infection of a eukaryotic cell, and the fact that a Tse2 hyper-secreting strain is attenuated in animal models of acute infection (Laskowski et al., 2004; Zolfaghar et al., 2006), suggests that the T6S apparatus can be highly discriminatory. In this regard, it is instructive to consider other secretion systems that have evolved from inter-bacterial interaction pathways. The type IVA and type IVB secretion systems are postulated to have evolved from a bacterial conjugation system ((Burns, 2003; Christie et al., 2005; Lawley et al., 2003). These systems have become efficient at eukaryotic cell intoxication, however measurements indicate that substrate translocation into bacteria occurs at a frequency of only ~1 × 10−6/donor cell (Luo and Isberg, 2004). In contrast, Tse2 targeting to bacteria by the H1-T6SS appears many orders of magnitude more efficient, as the donor strain in our assays is able to effectively suppress the net growth of an equal amount of recipient cells. The host adapted type IV secretion systems and the H1-T6SS represent two apparent extremes in the cellular targeting specificity of Gram-negative specialized secretion systems. Furthermore, they show that a high degree of discrimination can exist between pathways targeting eukaryotes and prokaryotes.

The physiologically relevant target bacteria of Tse2 and the H1-T6SS remains an open question. We have initiated studies to address the role of these factors in interspecies interactions, however we have not yet identified an effect. This may be because diffusible anti-bacterial molecules released by P. aeruginosa dominate the outcome of growth competitions performed under the conditions used in Figure 6 (Hoffman et al., 2006; Kessler et al., 1993; Voggu et al., 2006). In future studies designed to allow free diffusion of these factors, and thereby more closely mimic a natural setting, their role may be mitigated. Interestingly, all sequenced P. aeruginosa strains appear to encode orthologs of tse2 and tsi2. Additionally, we found the genes universally present within a library of 44 randomly selected CF patient clinical isolates (Figure S2). Despite these findings, it remains possible that Tse2-mediated inter-P. aeruginosa interactions could be relevant in a natural context. For instance, it may not be simply the presence or absence of the toxin or its immunity protein, but rather the extent and manner in which these traits are expressed that decides the outcome of an interaction. In prior investigations of clinical isolates, we noted a high degree of heterogeneity in H1-T6SS activation, as judged by Hcp1 secretion levels (Mougous et al., 2006; Mougous et al., 2007). The wild-type strain used in the current study does not secrete Hcp1, and in this background the H1-T6SS does not provide a growth advantage against an immunity-deficient strain (data not shown). However, the H1-T6SS activation state of many clinical isolates resembles the ΔretS background, and therefore these strains are likely capable of using Tse2 in competition with other bacteria. In this context, it is intriguing that tse and HSI-I expression are subject to strict regulation by the Gac/Rsm pathway (Figure 3E). Since this pathway responds to bacterial signals, including those of the sensing strain and other Pseudomonads (Lapouge et al., 2008), it is conceivable that cell-cell recognition could be an important aspect of Tse2 production and resistance.

The cell-cell contact requirement of H1-T6SS-dependent delivery of Tse2 suggest that the system could play an important role in scenarios involving relatively immobile cells, such as cells encased in a biofilm. The polyclonal and polymicrobial lung infections of patients with CF, wherein the bacteria are thought to reside within biofilm-like structures, is one setting where Tse2 could provide a fitness advantage to P. aeruginosa (Sibley et al., 2006; Singh et al., 2000). Intriguingly, P. aeruginosa is particularly adept at adapting to and competing in this environment, and studies have shown that it can even displace preexisting bacteria (D’Argenio et al., 2007; Deretic et al., 1995; Hoffman et al., 2006; Nguyen and Singh, 2006; CF Foundation, 2007). If Tse2 does play a key role in the fitness of P. aeruginosa in a CF infection, this could explain the elevated expression and activation of the H1-T6SS observed in P. aeruginosa isolates from CF patients (Mougous et al., 2006; Mougous et al., 2007; Starkey et al., 2009; Yahr, 2006).

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids and Growth Conditions

The P. aeruginosa strains used in this study were derived from the sequenced strain PAO1 (Stover et al., 2000). P. aeruginosa were grown on Luria–Bertani (LB) medium at 37°C supplemented with 30 μg ml−1 gentamicin, 300 μg ml−1 carbenicillin, 25 μg ml−1 irgasan, 5% w/v sucrose, 0.5 mM IPTG and 40 μg ml−1 X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) as required. Burkholderia thailandensis E264 and Escherichia coli BL21 were grown on LB medium containing 200 μg ml−1 trimethoprim, 50 μg ml−1 kanamycin, 0.2% w/v glucose, 0.2% w/v rhamnose and 0.5 mM IPTG as required. E. coli SM10 used for conjugation with P. aeruginosa was grown in LB medium containing 15 μg ml−1 gentamicin. Plasmids used for inducible expression include pPSV35, pPSV35CV, and pSW196 for P. aeruginosa (Baynham et al., 2006; Hsu et al., 2009; Rietsch et al., 2005), pET29b (Novagen) for E. coli, pSCrhaB2 (Cardona and Valvano, 2005) for B. thailandensis, and p426-GAL-L and p423-GAL-L for S. cerevisiae (Mumberg et al., 1995). Chromosomal fusions and gene deletions were generated as described previously (Mougous et al., 2006; Rietsch et al., 2005). See Supplemental Experimental Procedures for specific cloning procedures.

Secretome Preparation

Cells were grown in Vogel-Bonner minimal medium (VBMM) (Schweizer, 1991; Vogel and Bonner, 1956) containing 19 mM amino acids as defined in synthetic CF sputum medium by Palmer et al. 2007 (Palmer et al., 2007). We empirically determined that the presence of amino acids in VBMM was a requirement for H1-T6SS activity (data not shown). Overnight (o/n) cultures were synchronized by two rounds of subculturing to optical density 600 nm (OD600) 0.05. The final culture (20 ml) was grown to OD600 1.0, whereupon cells were removed by centrifugation (3,200 × g, 10 min) at room temperature and filtration of the resulting supernatant (0.22 μm filter, Millipore). Deoxycholate was added to 0.2 mg/ml and the solution was incubated for 30 min on ice. Trichloroacetic acid (TCA) was added to 10 % v/v, and precipitated proteins were recovered by centrifugation (16,000 × g, 30 min) at 4 °C and washed once with acetone.

Mass Spectrometry

Precipitated proteins were suspended in 100 μl of 6 M urea in 50 mM NH4HCO3, reduced and alkylated with dithiotreitol and iodoactamide, respectively, and digested with trypsin (50 : 1 protein : trypsin ratio). The resultant peptides were desalted with Vydac C18 columns (The Nest Group) following the manufacturer’s protocol. Samples were dried to 5 μL, resuspended in 0.1% formic acid/5% acetonitrile and analyzed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher) in triplicate. Data was searched using Sequest (Eng et al., 1994) and validated with Peptide/Protein Prophet (Keller et al., 2002). The relative abundance for identified proteins was calculated using spectral counting (Liu et al., 2004). See Supplemental Experimental Procedures additional MS procedures.

Preparation of Proteins and Western Blotting

Cell-associated and supernatant samples were prepared as described previously (Hsu et al., 2009). Western blotting was performed as described previously (Mougous et al., 2006), with the exception that detection of the Tse proteins required primary antibody incubation in 5% BSA in Tris-buffered saline containing 0.05% v/v Tween 20 (TBST). The GSK tag was detected using α-GSK (Cell Signaling Technologies).

Immunoprecipitation

Cells grown in appropriate additives were harvested at mid-log phase by centrifugation (6,000 × g, 3 min) at 4°C and resuspended in 10 ml of Buffer 1 (200mM NaCl, 20mM Tris pH 7.5, 5% glycerol, 2 mM dithiothreitol, 0.1% triton) containing protease inhibitors (Sigma) and lysozyme (0.2 mg ml−1). Cells were disrupted by sonication and the resulting lysate was clarified by centrifugation (25,000 × g, 30 min) at 4°C. A sample of the supernatant material was removed (Pre) and the remainder was incubated with 100 μl of α-VSV-G agarose beads (Sigma) for 2 hours at 4°C for. Beads were washed three times with 15 ml of Buffer 1 and pelleted by centrifugation. Proteins were eluted with SDS-PAGE loading buffer.

Fluorescence Microscopy

Mid-log phase cultures were harvested by centrifugation (6,000 × g, 3 min), washed with phosphate-buffered saline (PBS), and resuspended to OD600 5 with PBS containing 0.5 mM TMA-DPH (Molecular Probes). Microscopy was performed as described previously (Hsu et al., 2009). All images shown were manipulated identically.

Yeast Toxicity Assays

Saccharomyces cerevisiae BY4742 (MATα his3Δ 1 leu2Δ 0 lys2Δ 0 ura3Δ 0) was transformed with p426-GAL-L containing tse1, tse2, tse3, or the empty vector, and grown o/n in SC – Ura + 2% glucose (Mumberg et al., 1995). Cultures were resuspended to OD600 1.0 with water and serially diluted fivefold onto SC – Ura +2% glucose agar or SC – Ura +2% galactose + 2% raffinose agar. Plates were incubated at 30°C for 2 days before being photographed. The tsi2 gene was cloned into p423-GAL-L and transformed into S. cerevisiae BY4742 harboring the p426-GAL-L plasmid. Cultures were grown o/n in SC – Ura – His +2% glucose.

Growth Competition Assays

Overnight cultures were mixed at the appropriate donor-to-recipient ratio to a total density of approximately 1.0 × 108 CFU/ml in 5 ml LB medium. In each experiment, either the donor or recipient strain contained lacZ inserted at the neutral phage attachment site (Vance et al., 2005). This gene had no effect on competition outcome. Co-cultures were either filtered onto a 47-mm 0.2 μm nitrocellulose membrane (Nalgene) and placed onto LB agar or were inoculated 1:100 into 2 ml LB (containing 0.4% w/v L-arabinose, if required), and were incubated at 37°C with shaking. Filter-grown cells were resuspended in LB medium and plated on LB agar containing X-gal.

Cell culture and infection assays

HeLa cells were cultured and maintained in Dulbecco’s modified eagle medium (DMEM, Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS) and 100 μg ml−1 penicillin or streptomycin as required. Incubations were performed at 37°C in the presence of 5% CO2. Infection assays were carried out using cells seeded in 96-well plates at a density of 2.0 × 104 cells/well. Following o/n incubation, wells were washed in 1X Hank’s balanced salt solution and DMEM lacking sodium pyruvate and antibiotics was added. Bacterial inoculum was added to wells at a multiplicity of infection of 50 from cultures of OD600 1.0. Following incubation for 5 hours, the percent cytotoxicity was measured using the CytoTox-One assay (Promega).

Transient transfection, cell rounding assays, and flow cytometric analysis

HeLa cells were seeded in 24-well flat bottom plates at a density of 2.0 × 105 cells/well and incubated o/n in DMEM supplemented with 10% FBS. Reporter co-transfection experiments were performed using Lipofectamine according to the manufacturer’s protocol. Total amounts of transfected DNA were normalized using equal quantities of the GFP reporter plasmid (empty pEGFP-N1 (Clonetech)), one of the tse expression plasmids (pEGFP-N1-derived), and either a non-specific plasmid or the tsi2 expression plasmid where indicated. Cell rounding was quantified manually using phase-contrast images from three random fields acquired at 40X magnification. Prior to flow cytometry, HeLa cells were washed two times and resuspended in 1X PBS supplemented with 0.75% FBS. Analysis was performed on a BD FACscan2 cell analyzer and mean GFP intensities were calculated using FlowJo 7.5 software (Tree Star, Inc.).

Supplementary Material

01

Acknowledgments

We thank A. Rietsch, S. Dove, and P. Singh for critical insights and careful review of the manuscript, B. Traxler, G. Nester, and L. Hoffman for helpful discussions, B. kulasekara and H. kulasekara for assistance with microscopy, members of the Miller laboratory for technical advice, D. wozniak for sharing reagents, and members of the Harwood, Parsek, Singh, Scott, and Greenberg laboratories for sharing their insight, reagents, and support. This work was supported by grants to J.D.M. from the NIH (AI080609) and University of Washington Royalty Research Fellowship (RRF 4324).

Footnotes

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