Type 6 Secretion System–Mediated Immunity to Type 4 Secretion System–Mediated Gene Transfer

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Science  11 Oct 2013:
Vol. 342, Issue 6155, pp. 250-253
DOI: 10.1126/science.1243745

Bacterial Détente?

Type VI secretion systems (T6SS) correspond to dynamic intracellular organelles that are functionally analogous to contractile bacteriophage tails. The T6SS of several bacteria species have been found to be responsible for antagonistic behavior that likely reflects the translocation of toxic proteins (effectors) between cells. Pseudomonas aeruginosa is able to sense exogenous T6SS attack and assemble its own T6SS apparatus to launch a retaliatory attack aimed directly at the attacker. Now, Ho et al. (p. 250) describe how exogenous attack is sensed in a process that involves membrane disruption and suggest that the T6SS provides a general cellular defense mechanism against not only T6SS but also conjugative DNA elements delivered via the type IV secretion system involved in mating pair formation.


Gram-negative bacteria use the type VI secretion system (T6SS) to translocate toxic effector proteins into adjacent cells. The Pseudomonas aeruginosa H1-locus T6SS assembles in response to exogenous T6SS attack by other bacteria. We found that this lethal T6SS counterattack also occurs in response to the mating pair formation (Mpf) system encoded by broad-host-range IncPα conjugative plasmid RP4 present in adjacent donor cells. This T6SS response was eliminated by disruption of Mpf structural genes but not components required only for DNA transfer. Because T6SS activity was also strongly induced by membrane-disrupting natural product polymyxin B, we conclude that RP4 induces “donor-directed T6SS attacks” at sites corresponding to Mpf-mediated membrane perturbations in recipient P. aeruginosa cells to potentially block acquisition of parasitic foreign DNA.

Bacteria often exhibit antagonistic behaviors toward each other in microbial communities (1). One molecular mechanism mediating such behavior is the type VI secretion system (T6SS) (2). The T6SS is a widely conserved (3) dynamic multicomponent nanomachine structurally related to contractile phage tails (4, 5). Gram-negative bacteria use the T6SS to kill prokaryotic and eukaryotic prey cells through contact-dependent delivery of toxic effectors (6, 7). In P. aeruginosa, T6SS encoded by the H1-T6SS cluster (8) selectively targets T6SS+ bacteria that attack it by sensing these exogenous attacks and posttranslationally activating its own T6SS at the precise location of the initial assaults (9, 10). We previously hypothesized that the signal triggering the T6SS counterattack was the perturbation of the cell envelope (10). Thus, we wondered whether other systems capable of breeching the cell envelope would trigger a similar T6SS response. One system capable of delivering macromolecules across the envelopes of other Gram-negative cells is the type IV secretion system (T4SS) (11). This secretion system is associated with conjugative elements such as the broad-host-range, IncPα plasmid RP4 (12) as well as virulence elements in several bacterial species (13). T4SS-mediated DNA conjugation involves three sets of proteins: (i) the core structure and pilus components comprising the mating pair formation (Mpf) complex, (ii) the relaxosome complex, which initiates DNA transfer by binding to and nicking the origin of transfer, and (iii) a coupling protein that bridges the relaxosome and Mpf complexes (14). During conjugation, the pilus extends from donor cells to mediate close cell-cell contact with recipients, which allows transfer of the DNA-bound relaxosome components to occur (14).

If T4SS-mediated cell-cell interactions could trigger T6SS attack, donor cells of a heterologous conjugation-proficient T6SS species should be sensitive to killing by T6SS+ P. aeruginosa. Therefore, we determined whether carrying the RP4 plasmid affected survival of E. coli K12 strain MC1061 when grown in competition with P. aeruginosa. For consistency with previous studies (9, 10), a retS mutant with a transcriptionally up-regulated H1-T6SS locus was used. When mixed with P. aeruginosa, we recovered ~96% fewer viable E. coli cells carrying RP4 as compared with those lacking it (Fig. 1A). This difference was not observed for P. aeruginosa mutants that were T6SS (vipA) but was still observed in a triple mutant lacking the three known P. aeruginosa T6SS effectors Tse1, Tse2, and Tse3 (Fig. 1A) (7). Although a pppA mutant with a hyperactive but unregulated T6SS could slightly inhibit E. coli growth, there was no enhanced killing of E. coli cells carrying RP4 compared with those without it (Fig. 1A), and deletion of tagT, a gene critical for sensing exogenous T6SS attack (10), completely abolished E. coli killing (Fig. 1A). Furthermore, in a three-strain mixture containing RP4+ and RP4 E. coli with P. aeruginosa, only RP4+ E. coli were killed (Fig. 1B). Thus, T6SS-dependent killing of RP4+ E. coli involves the same attack-sensing mechanism implicated in the T6SS counterattack responses (10).

Fig. 1 Mpf induces a donor-directed T6SS attack in P. aeruginosa.

(A) Summary of competition assays between either MC1061 (gray) or MC1061 RP4 (black) and the indicated strains of P. aeruginosa. Reported are the numbers of colony-forming units (CFUs) of surviving E. coli. Data are mean ± SD with n = 4 to 8 independent replicates. (B) Summary of 3-strain competitions between MC1061, MC1061 RP4 ∆traG (Tra, but Mpf+), and P. aeruginosa. Surviving CFUs of each E. coli strain determined by plating on media selective for each strain (n = 6 independent replicates). (C) Map of the RP4 plasmid indicating positions of transposon insertions. Labels with two genes separated by a slash (for example, traE/traF) represent insertions into the intergenic region between the two genes. (D) Plot of T6SS activation efficiency versus conjugation efficiency for each transposon mutant. Details on the indicated clusters are available in the text and table S1. Efficiencies are scaled so that values for wild-type RP4 are 100% and the RP4 parent strain are 0%. The blue dot represents wild-type RP4. The lower limit of detection for our assay was ~200 conjugants; mutants for which the conjugation efficiency was below this number are reported as 0% in the graph.

We next determined the genetic requirements for the RP4-dependent induction of the P. aeruginosa T6SS donor–directed attack. RP4 was subjected to transposon mutagenesis and transformed into E. coli strain MC1061. Individual mutants were sequenced to determine transposon insertion sites (Fig. 1C). Conjugation efficiency into recipient E. coli strain MG1655 was then determined for each of these RP4 mutants, and T6SS activation efficiency was calculated from the survival rate of MC1061 E. coli with these mutant plasmids grown in competition with T6SS+ P. aeruginosa (table S1). Plotting the data for each mutant revealed several different phenotype clusters (Fig. 1D). Mutants in cluster 1 maintained wild-type levels of conjugation efficiency and induced T6SS killing at levels comparable with the wild-type plasmid. Most of these mutants were insertions in genes outside of the tra1 or tra2 loci, the exceptions being a disruption in the RP4 entry exclusion factor trbK (15) and a disruption of traE, a topoisomerase III homolog (16). Neither of these genes are required for the Mpf system or DNA transfer (table S1) (17). Mutants in cluster 2 were completely defective in their ability to transfer DNA and did not induce the T6SS donor–directed killing response in P. aeruginosa. All of these insertions disrupted genes encoding Mpf structural components (table S1). There were two outliers not quite in cluster 1 or 2 that were still able to transfer DNA but did not induce a T6SS response (table S1): insertions in trbH, a lipoprotein believed to connect the pilus to the core complex (14); and trbN, a periplasmic transglycosylase that remodels the donor peptidoglycan and is required for pilus synthesis (14). Similar transposon disruptions of homologs of trbH (18) and trbN (19) in heterologous T4SSs affect the formation and stability of the Mpf pili. Mutants in cluster 3 induced a greater donor-directed T6SS response than that of wild-type RP4 but were defective in DNA conjugation (Fig. 2B). These mutants included disruptions of relaxosome components traI and traJ as well as coupling protein traG (table S1). Like those in cluster 3, mutants in cluster 4 also induced more T6SS killing than did the wild type but exhibited no defect in conjugation. Although it remains unclear why cluster-3 and -4 mutants induce more efficient T6SS-mediated killing, it is clear that successful DNA transfer is not required to trigger a T6SS attack by P. aeruginosa. We next determined whether other conjugative plasmids were also able to induce donor-directed T6SS attack. IncN compatibility group plasmid pKM101 (20) induced a T6SS attack comparable with that of RP4 (Fig. 2A), whereas E. coli carrying the sex factor F plasmid was unaffected by T6SS+ P. aeruginosa (Fig. 2B). It is not known why the E. coli F factor cannot be successfully transferred into P. aeruginosa (21), but this observation suggests that T6SS activation correlates to some degree with whether the host range of a given plasmid includes P. aeruginosa.

Fig. 2 IncN but not IncF induces donor-directed T6SS attacks.

(A and B) Summary of E. coli survival after competition with T6SS+ (black bars) or T6SS (gray bars) P. aeruginosa. Data are mean ± SD, n = 3 independent replicates. (A) Competition assays between P. aeruginosa and E. coli MG1655 carrying no plasmid, RP4, or pKM101. pKM101 confers streptomycin resistance, so MG1655 rather than MC1061 was used. (B) Competition assays between P. aeruginosa PAO1 and E. coli MC1061 carrying no plasmid, RP4, RP4 hyper-inducer Tra Mpf+ mutant (∆traG), or Fʹ. Fʹ was confirmed to be functional by successfully mating into several different E. coli strains.

If the P. aeruginosa donor-directed T6SS attack could be triggered by the Mpf system of donor species, then this attack might suppress plasmid transfer into a population of T6SS+ P. aeruginosa cells. Accordingly, we measured the frequency with which the plasmid pPSV35 (22) could be transferred into T6SS+ or T6SS P. aeruginosa from the E. coli donor strain SM10 (23), which carries a chromosomally integrated RP4 plasmid. Because pPSV35 does not encode its own transfer machinery but can be mobilized by the SM10-encoded conjugation system (22), the frequency with which P. aeruginosa cells acquired pPSV35 reflects the efficiency at which this plasmid is transferred into but not between P. aeruginosa cells. When donor E. coli and recipient P. aeruginosa were mixed at a 1:1 ratio, we observed an ~86% decrease in conjugation efficiency into a T6SS+ strain as compared with its isogenic T6SS vipA mutant (Fig. 3A). This reduction in transfer efficiency did not match the observed magnitude of killing of RP4+ MC1061 (Fig. 1A), probably because of intrinsic differences in the ability of various donor strains to promote Mpf and T6SS activation with P. aeruginosa, which is similar to what we observed in our RP4 mutant analysis (Fig. 1D). P. aeruginosa mutants defective in the attack-sensing pathway genes tagT and pppA (10) also exhibited greater conjugation efficiency as recipient strains (Fig. 3A). Examination of mixtures of T6SS+ P. aeruginosa and E. coli RP4+ donor cells by means of fluorescence microscopy revealed rounding and blebbing of E. coli cells—a response that is typical of T6SS-mediated bacterial killing (Fig. 3B). Thus, inhibition of the conjugative transfer of pPSV35 was likely due to killing of E. coli cells through a donor-directed T6SS attack by P. aeruginosa.

Fig. 3 Donor-directed T6SS attack blocks heterologous transfer of DNA.

(A) The conjugation efficiency into different P. aeruginosa mutants. Data are mean ± SD, n = 7 independent replicates. (B) Representative field of cells containing a mixture of P. aeruginosa PAO1 ∆retS clpV1-gfp (green) and E. coli S17-1 RP4+ donor cells (nonfluorescent). E. coli cells exhibit cell rounding characteristic of T6SS-mediated killing (arrows). Larger magnification of rounding cells are shown in the insets.

The fact that multiple secretion systems can induce a T6SS counterattack suggested that the signal initiating this response really is a generalized disruption of the P. aeruginosa membrane. Accordingly, we asked whether polymyxin B—an antibiotic known to disrupt Gram-negative bacterial membranes by binding the lipid A component of lipopolysaccharides (2426)—could induce T6SS activity in P. aeruginosa. We used a P. aeruginosa strain carrying a ClpV1-GFP and fluorescent time-lapse microscopy to monitor T6SS organelle formation and dynamics (9, 10) after exposure to polymyxin B. Cells exhibited a sixfold increase in the average number of visible ClpV1-GFP foci per cell within 90 s of being spotted onto agar pads containing 20 μg/mL of polymyxin B (Fig. 4, A and C, and movie S1). After this increase in T6SS activity, most ClpV1-GFP foci disappeared over the next 3 min, with the remaining foci becoming nondynamic (Fig. 4A and movie S1). The loss of dynamics likely reflects consumption of intracellular adenosine 5′-triphosphate pools after prolonged exposure to polymyxin B intoxication. This increase in T6SS activity was not observed when cells were spotted onto agar pads lacking polymyxin B (Fig. 4, A and D, and movie S1). Additionally, this increase in ClpV1-GFP foci was not observed in tagT mutants even in the presence of polymyxin B (Fig. 4, A, E, and F; and movie S2), suggesting that the same attack-sensing pathway that senses T4SS and T6SS attacks is responding to this antibiotic and mediates activation of the T6SS.

Fig. 4 Activation of T6SS organelle formation in response to polymyxin B treatment requires TagT.

(A) Wild-type (WT) or tagT mutant (∆tagT) P. aeruginosa cells were imaged every 10 s starting immediately after being spotted onto agar pads containing 0 [untreated (UT)] or 20 μg/mL polymyxin B (PB). Total number of ClpV1-GFP foci was divided by the number of cells for each field of cells to determine the average number of foci per cell. Each curve represents the mean of 12 to 16 fields with 250 to 600 cells in each field ± SD. (B) Color scale used to temporal-color code ClpV1-GFP signal. (C to F) ClpV1-GFP localization was followed for 5 min and temporally color-coded.

These studies support a model in which the donor-directed T6SS attack response in P. aeruginosa likely involves detection of perturbations in the cell envelope caused by the invasive components of the T4SS conjugation machinery. T6SS may represent a type of bacterial “innate immune system” that can detect and attack invading infectious elements not by recognizing their molecular patterns [such as nucleic acid sequences, as do the clustered regularly interspaced short palindromic repeat (CRISPR) elements (27, 28); or unmethylated DNA, as do restriction enzymes (29)] but rather by recognizing “transfer-associated patterns” (TAPs), including membrane disruptions that occur during interactions with other cells deploying T6SS and T4SS translocation machines. Broad-host-range conjugative elements represent infectious bacterial “diseases” that can cause metabolic stress on their newly acquired hosts and thus represent a fitness burden to bacterial populations unable to combat their acquisition. The donor-directed T6SS attack paradigm may represent a strategy for suppressing the movement of horizontally transferred genetic elements in bacterial populations regardless of their signature molecular patterns (such as nucleic acid chemical structures or primary sequences).

Supplementary Materials

Materials and Methods

Table S1

References (30, 31)

Movies S1 and S2

References and Notes

  1. Acknowledgments: Supporting movies and table can be found in the supplementary materials. This work was supported by National Institute of Allergy and Infectious Diseases grants AI-018045 and AI-26289 to J.J.M.
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