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Type 6 Secretion Dynamics Within and Between Bacterial Cells

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Science  17 Aug 2012:
Vol. 337, Issue 6096, pp. 815
DOI: 10.1126/science.1222901

Abstract

The bacterial type 6 secretion system (T6SS) functions as a virulence factor capable of attacking both eukaryotic and prokaryotic target cells by a process that involves protein transport through a contractile bacteriophage tail-like structure. The T6SS apparatus is composed, in part, of an exterior sheath wrapped around an interior tube. Here, we report that in living cells the cytoplasmic adenosine triphosphatase called ClpV specifically recognizes the contracted T6SS sheath structure, causing its disassembly within seconds. ClpV imaging allowed spatial and temporal documentation of cell-cell interactions (termed T6SS dueling) that likely mark the location of repeated T6SS-mediated protein translocation events between bacterial cells.

The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction (13). In Vibrio cholerae, two proteins (VipA and VipB) build a phage tail sheathlike tubular structure in the cytosol of “predator” cells that is either extended or contracted (3). Contraction of the extended VipA/VipB sheath is thought to drive the T6SS spike and inner tube complex out of the effector or predator cell and into an adjacent target or “prey” cell (3). Disassembly of the cytoplasmic contracted sheath requires ClpV in vivo (3), a AAA+ adenosine triphosphatase (ATPase) that binds VipA/VipB tubules in vitro and can remodel these structures in the presence of ATP (4, 5). In Pseudomonas aeruginosa, ClpV1–green fluorescent protein (GFP) localizes to discrete foci that depend on T6SS function (6).

We used time-lapse fluorescence microscopy to follow ClpV localization in live V. cholerae 2740-80 cells. Functional ClpV–super folder GFP (sfGFP) and mCherry2 fusion proteins assembled at random times into short structures that disappeared in tens of seconds. In the ∆VipA background, ClpV was evenly distributed in cytosol, suggesting that the short ClpV structures were dependent on T6SS sheaths (fig. S1 and movie S1). We used functional VipA-sfGFP (3) and ClpV-mCherry2 fusions to image ClpV and T6SS sheaths simultaneously. Extended VipA-sfGFP–containing sheaths were not colocalized with ClpV-mCherry2, whereas contraction of a sheath led to immediate colocalization of ClpV-mCherry2 with the whole contracted sheath (Fig. 1 and movies S2 and S3). Half of the ClpV associated with the contracted sheath between 683 and 1273 ms (average = 952 ms, SD = 164 ms, n = 10; fig. S2 and movie S4). The disassembly of the contracted sheath required between 22 and 46 s (average = 32.5 s, SD = 6.1 s, n = 40), measured from the moment of contraction to the moment when both ClpV and VipA signals were no longer colocalized to one spot (movies S2 and S3).

Fig. 1

ClpV colocalizes with contracted sheath (arrows). A 3-μm-by-3-μm field of cells is shown. Scale bar in (A) is 1 μm and applies to (A) to (D). (A to C) V. cholerae ClpV-mCherry2 + pBAD24-VipA-sfGFP. (A) Merge of ClpV-mCherry2 and VipA-sfGFP signals. (B) ClpV-mCherry2 signal. (C) VipA-sfGFP signal. Additional frames and cells are shown in movies S2 and S3. (D) P. aeruginosa ΔretS/ClpV1-GFP; additional frames and cells are shown in movies S6 and S7.

The Tyr664→Ala664 (Y664A) mutation in the pore of ClpV blocks VipA/VipB disassembly but still allows binding of ClpV to VipB in vitro (4) whereas the Phe87→Arg87 (F87R) mutation of N-terminal domain of ClpV blocks VipB recognition in vitro (5). In vivo, ClpV-Y664A-mCherry2 was colocalized with VipA-sfGFP to short nondynamic structures that were likely contracted T6SS sheaths (fig. S3 and movie S5). In contrast, ClpV-F87R-mCherry2 was distributed uniformly in the cytosol with only VipA-sfGFP localized into contracted nondynamic sheaths (fig. S3 and movie S5). Localization of these ClpV mutants and the change in the dynamics of VipA-containing structures are consistent with published data (4, 5) and suggest that, in vivo, the N terminus of VipB is exposed on the surface of the contracted sheath just before its disassembly.

In P. aeruginosa, mutation of the regulatory gene retS allows expression of one of its T6SS loci (6). To assess the dynamics of T6SS in P. aeruginosa, we imaged a ClpV1-GFP fusion protein in a retS mutant (6) by time-lapse fluorescence microscopy. In contrast to V. cholerae, only a subset of P. aeruginosa cells actively formed and disassembled ClpV1-GFP–containing complexes during the observation period; the formation and dynamics of these structures required the VipA homolog PA0083 (fig. S1 and movies S6 and S7). ClpV1-GFP structures often assembled and disassembled repeatedly in apparently the same subcellular location (Fig. 1D and movie S7) indicating that, in contrast to V. cholerae (3), multiple T6SS apparatuses assemble in close proximity or, more likely, T6SS “base plate” components (3) are recycled by P. aeruginosa.

P. aeruginosa cells responded to T6SS activity occurring in a neighboring sister cell with an increase in their own T6SS dynamics (Fig. 1D and movies S6 and S7). Over time, the coincidence of T6SS activity between pairs of sister cells (termed T6SS dueling) became the dominant category of T6SS activity observable in the P. aeruginosa population (table S1 and fig. S4). Spatially concurrent T6SS activity could not be documented between V. cholerae sister cells because this species exhibited much higher levels of T6SS activity in nearly all cells (fig. S4). The spatial and temporal coincidence of T6SS activity in adjacent P. aeruginosa cells strongly suggests that a signal was being transferred between cells precisely at the position of the initial T6SS activity. The P. aeruginosa T6SS is thought to transfer peptidoglycan-hydrolyzing T6SS substrates into sister cells that express immunity proteins to their action (7). Perhaps cellular attack by a T6SS apparatus mediated by translocation of T6SS components (e.g., the spike/inner tube complex or effector proteins) into nearby adjacent sister cells induces local cell-envelope alterations [e.g., membrane perturbation, mild peptidoglycan hydrolysis, or protein phosphorylation (7, 8)] that trigger the formation of a T6SS apparatus in the vicinity of such alterations (fig. S5).

ClpV imaging provides evidence that P. aeruginosa likely recycles T6SS membrane base plate components and can sense T6SS activity in nearby cells. Because T6SS dueling events were spatially and temporally linked, they likely mark the exact location of T6SS translocation of protein components (e.g., VgrG and/or effector proteins) between cells. T6SS dueling may reflect social interactions between heterologous T6SS+ species that coexist in the same niche.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1222901/DC1

Materials and Methods

Figs. S1 to S5

Table S1

References (911)

Movies S1 to S7

References and Notes

  1. Acknowledgments: We thank T. G. Bernhardt and N. T. Peters for suggestions on the use of fluorescence microscopy resources and B. Ho and K. Roberts for helpful discussions. This work was supported by National Institute of Allergy and Infectious Disease grants AI-018045 and AI-26289 to J.J.M.
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