Research Article

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network

Thibault G. Sana, Christoph Baumann, Andreas Merdes, Chantal Soscia, Thomas Rattei, Abderrahman Hachani, Cerith Jones, Keiryn L. Bennett, Alain Filloux, Giulio Superti-Furga, Romé Voulhoux, Sophie Bleves
Dara Frank, Invited Editor, Eric J. Rubin, Editor
DOI: 10.1128/mBio.00712-15

ABSTRACT

Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction.

IMPORTANCE Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

INTRODUCTION

One of the key requirements for pathogenic bacteria to establish an infection and persist in an immunocompetent host is successful avoidance of innate defense mechanisms. A frequently used strategy for immune evasion employed by invasive bacteria is to direct their uptake into host cells that are not normally phagocytic, including epithelial and endothelial cells lining mucosal surfaces and blood vessels, respectively. Presumably, this invasion tactic ensures a protected cellular niche for the pathogen to replicate or persist, and in some instances, it can also allow transit into deeper tissues through tight epithelial cell barriers. The most common way to induce phagocytosis involves the subversion of the actin remodeling machinery to manipulate the cytoskeleton into producing actin-rich cell surface projections specifically promoting the engulfment of the adherent bacteria (1, 2). Although most invasive bacteria utilize host actin for entry, some also appear to modulate the dynamics of host microtubules (3). Examples include enteropathogenic and enteroinvasive Escherichia coli (4), Haemophilus influenzae (5), Campylobacter jejuni, and Citrobacter freundii (6), Neisseria gonorrhoeae (7), or Burkholderia cepacia complex (BCC) (8). Although microtubules are not involved in the internalization processes, such as phagocytosis, treatment of epithelial cells with microtubule-destabilizing agents like colchicine or nocodazole decreases the number of internalized bacteria of these species. However, the molecular basis of microtubule-dependent invasion is still poorly understood.

Pseudomonas aeruginosa is an opportunistic bacterium of humans leading to various infections in immunocompromised people, including bacteremia, sepsis, pneumonia, and wound and skin infections (9). This pathogen is also responsible for chronic lung infections and lethal consequences in patients with cystic fibrosis. While considered an extracellular pathogen, P. aeruginosa is able to enter nonphagocytic cells such as epithelial and endothelial cells (10, 11). Different isolates vary in their internalization efficiency into cultured mammalian cells (12). Among them, the invasive strains, which are the most common in P. aeruginosa species, possess the ExoS effector, while the cytotoxic and hence noninvasive ones lack exoS and instead encode the acute cytotoxin ExoU, which can quickly kill cells (13–15). However, this ability of internalization has been maintained over the course of P. aeruginosa evolution, indicating a fundamental role in the infection, notably in cornea invasion (16). P. aeruginosa preferentially infects damaged epithelial tissues and exploits the epithelial cell polarization machinery (11, 13, 17, 18). In this process, the binding of the bacterium to the cell surface activates a central host-signaling molecule, phosphatidylinositol 3-kinase (PI3K), required for synthesis of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and for activation of a downstream effector, the Ser/Thr kinase Akt (19). The result is that the bacterium transforms apical into basolateral membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier.

We have demonstrated that among the various factors facilitating internalization of P. aeruginosa, the H2 type VI secretion system (H2-T6SS) is involved in the uptake of the bacterium by eukaryotic cells (20). An H2-T6SS mutant indeed displays a significant decrease in HeLa cell invasion and, concomitantly, a reduced Akt phosphorylation in infected lung epithelial cells. Recently, Jiang and colleagues showed that the H3-T6SS also mediates P. aeruginosa PAO1 uptake and that H2-T6SS and H3-T6SS can compensate for each other under certain growth conditions (21). Two phospholipases D, PldA (Tle5) and PldB, which depend on the H2-T6SS and H3-T6SS, respectively, are involved in Akt binding. The type VI secretion machinery allows Gram-negative bacteria to interact with either eukaryotes or bacteria and deliver effector proteins into target cells upon contact (22–25). The T6SS puncturing device is structurally related to the bacteriophage contractile tail and includes the hemolysin-coregulated protein (Hcp) and valine glycine repeat G (VgrG) proteins. A trimer of VgrG proteins may fulfill the role of the phage T4 (gp27/gp5)3 complex at the tip of the tail (26). Some of the T6SS machineries, like those of Vibrio cholerae (27–29) and P. aeruginosa (20, 21, 30), permit interaction with both eukaryotic and bacterial target cells. PldA and PldB are even transkingdom effectors since they target both prokaryotic and eukaryotic cells; each of them triggers killing of bacterial competitors and internalization of P. aeruginosa into nonphagocytic cells (21, 30, 31). The first discovered antieukaryotic T6SS effectors belong to a subclass of VgrG proteins, called evolved VgrGs, containing C-terminal extensions with specialized intracellular functions. The C-terminal extension, an actin cross-linking domain in VgrG1 of V. cholerae (27, 32) or an ADP-ribosyl transferase domain in VgrG1 of Aeromonas hydrophila (33), promotes a cytotoxic effect toward eukaryotic host cells by disrupting the actin cytoskeleton. These evolved VgrGs have a dual function, playing a structural role in the puncturing device as well as an effector function (34). The second type of antieukaryotic T6SS effector is an independent toxin targeted to the host cell. The N-terminal pleckstrin homology domain of VasX from V. cholerae may be implicated in binding the host membrane lipids and thus in the interference with host phosphoinositide metabolism and signaling (35). Likewise PldA and PldB bind Akt to promote P. aeruginosa internalization (21).

Here we investigated which H2-T6SS-related VgrG protein enables P. aeruginosa strain PAO1 to enter epithelial cells. We showed that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubule components, including the γ-tubulin ring complex (γTuRC), which precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered a novel target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

RESULTS

VgrG2a and VgrG2b are important for P. aeruginosa internalization.While the majority of T6SS loci contain in their immediate proximity hcp and vgrG genes (36), these are absent at the vicinity of the P. aeruginosa strain PAO1 H2-T6SS gene cluster (Fig. 1A). Based on phylogenetic clustering, two sets of orphan Hcp and VgrG proteins have been proposed as likely candidates to represent the two proteins required for the H2-T6SS function (36–38). These are proteins encoded by the hcp2a-vgrG2a (PA1512-PA1511) and hcp2b-vgrG2b (PA0263-PA0262) loci and may be part of larger operons (Fig. 1A). They may be constituents of the so-called “vgrG islands” (36), very likely acquired by horizontal gene transfer and frequently linked to genes encoding type VI lipase effector (Tle) with putative a GxSxG catalytic motif and potential lipase activity (30). In the vgrG2a island, the linked product of PA1510 has a putative PGAP1-like domain (PF07819) and belongs to the Tle4 family (30). In the vgrG2b island, the PA0260-encoded protein has an α-β hydrolase domain (PF00561) and has been classified in the Tle3 family (30). The products of PA1509 and PA0259 have been proposed to be the putative immunity proteins of Tle4 and Tle3, respectively (30). The two Hcp2 proteins are 100% identical, while the VgrG2a and VgrG2b proteins are not identical in size but share 93% identity over their first 828 residues (Fig. 1B). The N-terminal 581 residues of these two proteins contain the VgrG domain, homologous to (gp27)3-(gp5)3 phage-tail proteins, which is followed by a domain of unknown function (DUF2345) present in many VgrG proteins. VgrG2b contains an extended C-terminal extension with a putative zinc-dependent metallopeptidase domain (PS00142) and has been thus considered an evolved VgrG (32).

FIG 1 
FIG 1 

VgrG2a and VgrG2b are likely components of the H2-T6SS machinery. (A) vgrG2a and vgrG2b island organization. The numbers on the map indicate base pair numbers in millions. The genes are labeled with the given name (i.e., vgrG2a) or are indicated by their annotation number (e.g., PA0261). (B) VgrG2a and VgrG2b domain architecture. The first 581 residues carry the VgrG domain, consisting of the phage GPD (phage late control gene D protein, PF05954), phage base V (phage-related baseplate assembly protein, PF04717), and T6SS Vgr (putative type VI secretion system Rhs element, PF13296) domains, homologous to gp27 and gp5 phage tail proteins. This is followed by a conserved domain of uncharacterized proteins (DUF2345, PF10106) present in many VgrG proteins. VgrG2b possesses an additional C-terminal extension of 186 residues, with a putative zinc-dependent metallopeptidase pattern (LFIHEMTHVW signature, PS00142). (C) Bacterial invasion (gentamicin protection assays) of HeLa cells upon 3 h of infection at an MOI of 10 with P. aeruginosa strains grown at the transition between the exponential and stationary phases in Trypticase soy broth (TSB) at 37°C. The percentage of invasion of PAO1ΔclpV2, PAO1ΔvgrG2a, PAO1ΔvgrG2b, or PAO1ΔvgrG2a ΔvgrG2b was normalized to the WT PAO1 strain, which represents an average of 1.3 × 105 CFU of internalized bacteria per well (2.5 × 105 HeLa cells). Each experiment was performed a minimum of three times in triplicate. The error bars indicate standard deviations. ***, P ≤ 0.001.

To demonstrate a functional connection between H2-T6SS and these Hcp2-VgrG2 orphans, we took advantage of the role of the H2-T6SS machinery in P. aeruginosa internalization into nonphagocytic cells (20). We paid special attention to cultivating bacteria in the exponential phase before infecting HeLa cells (20) since H3-T6SS can take over H2-T6SS function in internalization when cells are grown in the stationary phase (21). This growth-phase-dependent compensation is explained by the expression patterns of the two gene clusters. Quorum sensing indeed induces the two gene clusters in the exponential phase, but whereas H2-T6SS expression is maximal during the log- to stationary-phase transition, H3-T6SS is efficiently expressed later in the stationary phase (39). As expected, upon P. aeruginosa infection of HeLa cells, only a few of the PAO1ΔclpV2 bacteria, an H2-T6SS-defective strain, were internalized in comparison to the wild-type (WT) PAO1 strain (Fig. 1C) (20, 21). The same impairment (about 75% decrease) was observed for the PAO1ΔvgrG2a, PAO1ΔvgrG2b, and PAO1ΔvgrG2aΔvgrG2b mutant strains, whereas the adherence of the mutant strains was not affected (see Fig. S1A in the supplemental material). The requirement of VgrG2a, VgrG2b, and the H2-T6SS machinery for the internalization was confirmed in a more physiologically relevant cell line, A549 human alveolar epithelial cells (see Fig. S1B). This demonstrates that internalization of P. aeruginosa requires a functional H2-T6SS and the two vgrG2 products (Fig. 1A and B). These results show a functional link between VgrG2a, VgrG2b, and the H2-T6SS machinery and identify VgrG2a as a component and the evolved VgrG2b as a very likely cognate effector.

Figure S1 

(A) Adherence of the PAO1ΔvgrG2a, PAO1ΔvgrG2b, and PAO1ΔvgrG2a ΔvgrG2b mutant strains on HeLa cells. The percentage of adherence of each mutant strain was normalized to WT PAO1 (black bar). Error bars represent standard deviations. All assays were performed a minimum of three times in triplicate. (B) Results from standard bacterial invasion assays in A549 cells upon infection with P. aeruginosa strains grown at the transition between exponential and stationary phases in TSB at 37°C. The percentages of invasion of PAO1ΔclpV2, PAOΔvgrG2a, PAO1ΔvgrG2b, and PAO1ΔvgrG2a ΔvgrG2b were normalized to the WT PAO1 strain, which represents an average of 4.8 × 105 CFU of internalized bacteria per well (2.5 × 105 A549 cells). Each experiment was performed a minimum of three times in triplicate. The error bars indicate standard deviations. *, P ≤ 0.05. Download Figure S1, PDF file, 0.2 MB.
Copyright © 2015 Sana et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

VgrG2b is delivered into host cells by the H2-T6SS machinery.We used confocal laser scanning microscopy to investigate whether VgrG2b is delivered into epithelial cells upon P. aeruginosa infection. In order to differentiate between the two VgrG2s when using immune detection, an antiserum specific to the VgrG2b C-terminal extension was developed (see Fig. S2A and B in the supplemental material). The examination of multiple individual cells revealed the presence of VgrG2b within some HeLa cells infected with the WT PAO1 strain (VgrG2b detection in 17 cells out of ≅600 observed cells) that are absent in the uninfected cells (Fig. 2A and B). When observed, the VgrG2b signals were not evenly distributed throughout the infected cells but instead were present as foci within the cytoplasm. On average, we observed one VgrG2b fluorescent dot per infected cell and in 3 cells more than one spot. Image acquisition at various focal levels of the epithelial cells confirmed that VgrG2b signal was within the infected cells (see Fig. S2C). Importantly, the VgrG2b intracellular localization was dependent on a functional H2-T6SS machine since foci were not detected upon infection with a clpV2 mutant (in no cell out of ≅600 observed cells) (Fig. 2), while VgrG2b was readily detected in the mutant bacteria (see Fig. S2D, left panel). Complementation in trans of the clpV2 mutation restored intracellular detection (in 13 cells out of ≅600 observed cells) (Fig. 2A and B). The very low level of intracellular VgrG2b (≈2.5%) might reflect the low level of internalized P. aeruginosa that we and others have previously observed (≈1.7% [12, 20]).

Figure S2 

Immunodetection of VgrG2b in P. aeruginosa (A) or in HEK293 cells (B) with the anti-VgrG2b C-terminal antiserum. Bacteria were grown at the transition between the exponential and stationary phases. PAO1 harboring pSBC63 overproduces VgrG2bHis upon induction of expression with 0.5% arabinose (PAO1 VgrG2b++). The samples from ectopic production of VgrG2aHA-Strep and VgrG2bHA-Strep in HEK293 cells are those of Fig. 3A. The equivalent of an optical density at 600 nm (OD600) of 0.1 U was loaded into each lane. Molecular mass markers are indicated in kilodaltons on the left. (C) Immunodetection of VgrG2b in HeLa cells infected with the WT PAO1 strain. Six images acquired at various focal levels of the epithelial cells (single Z-sections) are presented. VgrG2b and γ-tubulins 1 and 2 were labeled with anti-VgrG2b (1/500) or anti-GCP1-2 (1/1,000) antisera and detected with a secondary anti IgG antibodies conjugated to Cy3 and Cy5, respectively. VgrG2b is labeled in green, and γ-tubulins 1 and 2 are labeled in red. (D) Intrabacterial immunodetection of VgrG2bV5 and VgrG2aV5 with the anti-V5 antibodies. Bacteria were grown at the transition between the exponential and stationary phases (around 5 h at 37°C in TBS). The equivalent of 0.1 U at OD600 was loaded into each lane. Molecular mass markers are indicated in kilodaltons on the left. (E) Immunodetection assay of VgrG2aV5 in HeLa cells infected with the WT PAO1 strain. VgrG2aV5 was labeled with anti-V5 antiserum and detected with a secondary anti-IgG antibodies conjugated to Cy3. Representative images of individual cells after counting of 600 cells from four independent experiments are presented. Download Figure S2, PDF file, 0.8 MB.
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FIG 2 
FIG 2 

VgrG2b is delivered in the host cell upon P. aeruginosa infection. Shown are results from confocal laser-scanning microscopy of HeLa cells infected or not (uninfected) with P. aeruginosa PAO1 (WT) and the mutant strains indicated (A). The clpV2 mutation was complemented by expression in trans of clpV2 from the pHM49 plasmid (ΔclpV2 comp). VgrG2b was labeled with VgrG2b antiserum and detected with secondary anti-IgG antibodies conjugated to Cy3. Representative images of individual cells after counting of 600 cells from four independent experiments are presented. The black and red numbers indicate the number of cells in which VgrG2b is detected out of the total cell numbers observed. When indicated, 10 µg/ml of colchicine was added 30 min before HeLa cell infection. Scale bar, 5 µm. A graphical representation of the number of VgrG2b foci observed after counting of 600 cells is shown in panel B. ***, P ≤ 0.001 by χ2 test.

Moreover, the infection with a vgrG2a mutant that produces VgrG2b (see Fig. S2A and D, left panel, in the supplemental material) did not allow any intracellular immunodetection of VgrG2b, suggesting that VgrG2a may be required for VgrG2b delivery into host cells (Fig. 2A and B), which is in line with its involvement as an H2-T2SS component. As we have not ruled out that VgrG2a can also be injected, a chromosomally encoded VgrG2aV5 translational fusion was engineered in order to specifically detect VgrG2a. Whereas VgrG2aV5 was synthesized by P. aeruginosa (see Fig. S2D, right panel), we have been unable to detect it in infected cells (see Fig. S2E). This suggests either that VgrG2a is not targeted to the eukaryotic host cell, or the technique we used is not sensitive enough to detect VgrG2a translocation. The VgrG2a-dependent delivery of VgrG2b is in agreement with the heteromultimeric complexes formed by VgrGs (32, 38), although we have not been able to observe multimeric complexes containing VgrG2a and VgrG2b. Nevertheless this observation constitutes another piece of evidence that the orphan vgrG2a and vgrG2b islands are linked to the H2-T6SS cluster (Fig. 1A).

Altogether these data show that upon contact with the target cells, VgrG2b is delivered into the host cytoplasm by the H2-T6SS machinery involving VgrG2a. This is fully in agreement with the phylogenetic placement of VgrG2b as an evolved VgrG, an effector with putative activity in the infected mammalian cells.

An in vivo interactome approach identifies γTuRCs as partners of VgrG2b.In order to identify the host intracellular partners for VgrG2b, we made use of a tandem affinity purification-mass spectrometry (TAP-MS) procedure (40, 41). First a HEK293 cell line producing VgrG2bHA-Strep upon doxycycline induction was engineered (Fig. 3A and B). Moreover, to be able to exclude proteins copurifiying in a nonspecific manner, a cell line producing hemagglutinin-streptavidin (HA-Strep)-tagged green fluorescent protein (GFP) was used. Two-step tandem affinity purification for stringent enrichment of interacting proteins was performed (41). The analysis indicated that the TAP-tagged VgrG2b bound the α-tubulins (α-TUB1C and α-TUB1B) and β-tubulin, the microtubule subunits, which were also unspecifically recovered to a lesser extent with the control GFP (Fig. 3C; see Fig. S3 in the supplemental material). Interestingly the TAP-tagged VgrG2b also copurified specifically the full complement of proteins constituting the γ-tubulin complexes, such as γ-tubulin (GCP1), GCP2, and GCP3 of the γ-tubulin small complex(γTuSC), as well as GCP4, -5, and -6, NME7, and Mozart2B of the γ-tubulin ring complex (γTuRC) (Fig. 3C; see Fig. S3). The γ-tubulin complexes regulate microtubule nucleation, providing spatial and temporal control over the initiation of microtubule growth (42). In several animal cells, these nucleation sites are the centrosomes, which are organized around a pair of centrioles and serve as the central anchoring point for microtubules within the cell.

Figure S3 

Table summarizing all data obtained on peptide enrichment in 2 technical repeats each of 2 biological repeats each of 3 independent pulldowns. Download Figure S3, PDF file, 0.2 MB.
Copyright © 2015 Sana et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

FIG 3 
FIG 3 

VgrG2b interacts with γ-tubulin complexes in host cells. Immunodetection of ectopically produced VgrG2aHA-Strep and VgrG2bHA-Strep in HEK293 cells by Western blotting (A) or confocal microscopy (B). Doxycycline (1 µg/ml) was added during 24 h (A) or 16 h (B) for the induction of vgrG2aHAStrep and vgrG2bHAStrep. In panel A, VgrG2 proteins are detected with anti-VgrG2a antiserum, and α-tubulin immunodetection is used as a loading control. Molecular masses in kilodaltons are indicated on the left. In panel B, VgrG2 proteins are immunostained in green with anti-HA antiserum and α-tubulin in red. Scale bar, 5 µm. Proteins identified specifically in VgrG2b pulldowns are shown in panel C. Shown are average peptide counts (avPC) and average spectral counts (avSC) reflecting enrichment of proteins from two independent experiments, each processed in two technical replicates measured by LC-MS/MS. A full table including all detailed MS measurements is provided in Fig. S3 in the supplemental material. The most widely accepted model for the mechanism of γTuRC-based nucleation is presented and based on that of Kollman et al. (42). (D) Anti-HA coimmunoprecipitation assay in HEK293 cells producing VgrG2aHA-Strep or VgrG2bHA-Strep. Shown is immunodetection of VgrG2a/2bHA-Strep with anti-HA antiserum, GCP4 with anti-GCP4, and γ-tubulin with anti-GCP1 in cell lysate and nonbound fraction and immunoprecipitate (IP). Molecular masses in kilodaltons are indicated on the left.

As a specific negative control, the TAP-MS procedure was also applied to HEK293 cells producing the closely related VgrG2aHA-Strep (Fig. 3A and B). This did not lead to the copurification of any specific proteins (Fig. 3C and Fig. S3), whereas VgrG2aHA-Strep was much more produced than VgrG2bHA-Strep (Fig. 3A), suggesting that VgrG2b rather than VgrG2a has specificity for eukaryotic host interactions. Furthermore, this confirms the specificity of the detected VgrG2b interactions and suggests that the interaction with γ-tubulin complex proteins is likely mediated by the C-terminal extension of VgrG2b, which is absent in VgrG2a (Fig. 1B). It is also noteworthy that the spatial distributions of the two TAP-tagged VgrG2s produced in the HEK293 cell line are very different. Whereas VgrG2aHA-Strep had a diffuse localization throughout the cytoplasm (Fig. 3B), VgrG2bHA-Strep was observed as several foci, similar to the pattern seen following infection of cells with P. aeruginosa (Fig. 2A). This focal accumulation may reflect the targeting of VgrG2b to an organelle or a cellular complex.

To confirm the specific VgrG2b interaction with the γ-tubulin complex and exclude an indirect interaction through microtubules, we performed coimmunoprecipitation from HEK293 cell lysates under microtubule-depolymerizing conditions (43, 44) (Fig. 3D). The anti-HA antibodies specifically coprecipitated GCP4 and γ-tubulin (GCP1) from γTuRC, together with VgrG2bHA-Strep. Although VgrG2aHA-Strep was efficiently precipitated by the anti-HA antibodies, none of the tested proteins of the γ-tubulin complex coimmunoprecipitated. This suggests a role of the C-terminal extension of VgrG2b absent in VgrG2a in the interaction with the γTuRC, and this is independent of the microtubules themselves since microtubules are not polymerized under the conditions used in this experiment.

The T6SS-dependent internalization of P. aeruginosa is mediated by both microtubule and actin cytoskeleton.Since VgrG2b interacts with microtubule components, we next examined the requirement of microtubules for the H2-T6SS-dependent uptake of P. aeruginosa by epithelial cells. Although many pathogenic bacteria modulate the actin cytoskeleton of eukaryotic cells to facilitate the invasion of epithelial cells, some pathogens are capable of modulating the dynamics of host microtubules as well. Few studies have examined the requirement for microtubules during P. aeruginosa PAO1 strain internalization, and it seems uncertain since it is independent for corneal epithelial cell infection (18) and dependent for middle ear epithelial cells (45). Hence we studied P. aeruginosa uptake by HeLa cells in the presence of colchicine or nocodazole, two microtubule-destabilizing agents. Colchicine inhibited P. aeruginosa uptake in a dose-dependent manner (Fig. 4A). Importantly, the drug affected neither viability nor growth of the bacteria (data not shown) and moreover did not influence bacterial adherence (see Fig. S4A in the supplemental material). A similar decrease in cell invasion was observed following pretreatment of HeLa cells with various concentrations of nocodazole (see Fig. S4B). However, as expected from the literature (12, 18, 46), the internalization still requires the actin cytoskeleton since treatment with cytochalasin D (20 µM), which causes microfilament depolymerization, decreased P. aeruginosa uptake by HeLa cells (see Fig. S4C).

Figure S4 

Colchicine treatment does not impact adherence of P. aeruginosa (A). Shown is the percentage of adherence of PAO1 in the presence of colchicine (10 µg/ml) (white bar) normalized to PAO1 in absence of colchicine (black bar). Error bars represent standard deviations. All assays were performed a minimum of three times in triplicate. Nocodazole (B) and cytochalasin D (C) prevent H2-T6SS dependent internalization of P. aeruginosa. Shown is internalization of P. aeruginosa by HeLa cells with 1, 5, 10, 20, and 50 µg/ml of nocodazole (B) or 20 µg/ml of cytochalasin D (C) added (+) 1 h before infection with WT P. aeruginosa PAO1 for 3 h at an MOI of 10. As these inhibitors were dissolved in dimethyl sulfoxide (DMSO), HeLa cells treated with DMSO before infection serve as a control. ***, P ≤ 0.001. ns, not significant. Download Figure S4, PDF file, 0.2 MB.
Copyright © 2015 Sana et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

FIG 4 
FIG 4 

Colchicine prevents H2-T6SS-dependent internalization of P. aeruginosa. (A) Internalization of P. aeruginosa by HeLa cells with 0, 0.01, 0.1, 1, 10, and 50 µg/ml of colchicine added 30 min before infection with WT P. aeruginosa PAO1 for 3 h at an MOI of 10. The percentage of invasion was normalized to PAO1 in the absence of colchicine. (B) Internalization of different P. aeruginosa PAO1 mutants (at an MOI of 10) by HeLa cells with (+) or without (−) 10 µg/ml of colchicine added 30 min before infection for 3 h. The percentage of invasion was normalized to PAO1, which represents an average of 1.3 × 105 CFU of internalized bacteria per well (2.5 × 105 HeLa cells). Each experiment was performed a minimum of three times in triplicate. The error bars indicate standard deviations. ***, P ≤ 0.001.

To determine if P. aeruginosa delivers VgrG2b in host cells from the outside or once the bacterium has been internalized, we studied the VgrG2b foci in HeLa cells pretreated with colchicine at 10 µg/ml, a concentration inhibiting internalization (Fig. 4A). Since VgrG2b was still observed inside infected HeLa cells in the presence of colchicine (in 16 cells out of ≅600 observed cells [Fig. 2]) and while P. aeruginosa remained extracellular (Fig. 4A), we concluded that VgrG2b injection by the H2-T6SS machinery precedes the internalization of the bacterium. However, by using confocal microscopy, we have been unable to detect both VgrG2b and entering or intracellular bacteria. VgrG2b delivery by extracellular bacteria is thus a separate step from P. aeruginosa uptake.

We further hypothesized that if P. aeruginosa internalization triggered by the H2-T6SS machinery relies exclusively on a VgrG2b-mediated modulation of the microtubule cytoskeleton, colchicine treatment would not have any additional effect in an H2-T6SS mutant background (Fig. 4B). We indeed observed that the internalization defect of several H2-T6SS mutants (ΔclpV2, ΔvgrG2a, ΔvgrG2b, or ΔvgrG2aΔvgrG2b) is not increased by colchicine treatment of HeLa cells. We also observed that use of colchicine at a concentration of 10 µg/ml results in the same magnitude of internalization impairment (around 75%) as that in a strain with a mutation in the H2-T6SS. Based on these data, we propose that the H2-T6SS-dependent translocation of VgrG2b precedes and facilitates P. aeruginosa entry by microtubules and still requires actin microfilaments.

Bypassing the injection step by intracellular production of VgrG2b.We next investigated whether intracellular production of VgrG2b in the host prior to infection could boost the internalization of P. aeruginosa, even if other factors mediating P. aeruginosa uptake can be limiting (i.e., adhesins or PldA). We took advantage of the stable cell line overproducing VgrG2bHA-Strep and performed infection of these HEK293 cells with P. aeruginosa (Fig. 5). The induction of vgrG2bHA-Strep expression by doxycycline leads to an approximately 3-fold increase in the number of WT bacteria recovered in HEK293 cells after infection (compare bars 1 and 2 in Fig. 5). This effect is specific to VgrG2b since induction of vgrG2aHA-Strep expression in HEK293 cells had no effect on uptake (Fig. 5, bar 14). Moreover, it is still dependent on microtubules since colchicine decreased the entry (4.3-fold) (compare bars 2 and 4 in Fig. 5).

FIG 5 
FIG 5 

Bypassing the injection step. HEK293 cells overproducing (+ doxycycline) or not (− doxycycline) VgrG2bHA-Strep or VgrG2aHA-Strep were infected by different strains of P. aeruginosa in the presence (+) or not (−) of 10 µg/ml colchicine. Doxycycline (1 µg/ml) was added during 16 h for the induction of vgrG2aHA-Strep and vgrG2bHA-Strep. The cells were then infected during 3 h at a MOI of 10. The percentage of invasion was normalized to PAO1, which represents an average of 2.8 × 105 CFU of internalized bacteria per well (2.5 × 105 HEK293 cells). Each experiment was performed a minimum of three times in triplicate. The error bars indicate standard deviations. ***, P ≤0.001; *, P ≤0.05. ns, not significant.

To test if the intracellular pool of VgrG2b is sufficient to promote entry of bacteria impaired in internalization, we performed infections with invasion-deficient P. aeruginosa mutants lacking H2-T6SS or VgrG2b. Indeed both of these mutant strains were efficiently taken up by the HEK293 cells (bars 6 and 10 in Fig. 5), the intracellular production of VgrG2b increasing their uptake almost 2.8 times while maintaining dependency on microtubules (bars 8 and 12 in Fig. 5).

Altogether these data show that the intracellularly produced VgrG2b, presumably through interaction with tubulins and γTuRCs, can promote a microtubule-dependent entry of extracellular bacteria and even of bacteria that are normally impaired in invasion. This is another strong piece of evidence that VgrG2b delivery and P. aeruginosa uptake are two distinct steps.

DISCUSSION

A number of extracellular pathogens, including C. jejuni, C. freundii, H. influenzae, and B. cepacia complex, are able to invade nonphagocytic cells by modulating the dynamics of host microtubules. The basic mechanism of this important virulence-enhancing strategy is poorly understood. Here we demonstrated that upon infection by P. aeruginosa, the bacterial effector VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with the α- and β-tubulins and γTuRC. This interaction facilitates the uptake of the bacterium, a process dependent on microtubule dynamics since colchicine and nocodazole, two microtubule-destabilizing agents, impaired the internalization. Importantly P. aeruginosa internalization still involves the actin cytoskeleton. We thus provide a key insight into the self-promoted invasion process by showing that a bacterial effector is delivered into the host cells from extracellular bacteria to target microtubule subunits and in particular γTuRC, a multiprotein complex that promotes nucleation of microtubules.

Two orphan VgrGs, VgrG2a and VgrG2b, were experimentally validated as belonging to the H2-T6SS machine. The main difference between the two proteins is the extreme C-terminus extension of VgrG2b, which is absent in VgrG2a (Fig. 1B). The strains lacking either of one of the VgrGs or the H2-T6SS displayed the same internalization impairment by HeLa or A549 cells (Fig. 1C; see Fig. S1B in the supplemental material), which confirms a connection between the effectors and this particular T6SS machine despite the absence of a physical linkage between their respective genes (Fig. 1A). However, it seems that only VgrG2b is delivered into host cells upon infection of epithelial cells (Fig. 2) and is able to interact with the α- and β-tubulins and γTuRCs (Fig. 3C and D; see Fig. S3 in the supplemental material). The focal organization of VgrG2b into host cells (Fig. 2A and 3B) and the fact that VgrG2b binds γ-tubulins under microtubule-depolymerizing conditions (Fig. 3D) indicate that the interaction of VgrG2b with the γTuRC is α- and β-tubulin independent and probably direct. However, the interactants of VgrG2b within the γTuRC still remain to be identified. The evolved VgrG2b therefore fulfills a dual function as a structural component of the secretion apparatus and a true effector binding γTuRC, whereas VgrG2a may be considered only as a structural VgrG of the H2-T6SS puncturing device. Such bifunctional VgrGs are also recovered in V. cholerae, where VgrG1 cross-links actin of the target cell through its C-terminal extension (32) and VgrG3 degrades the peptidoglycan of neighboring bacteria (28, 29, 34).

The work reported here addresses the role of bacterial and host cellular factors during P. aeruginosa internalization and describes a role for VgrG2b in this process through its α- and β-tubulin-independent interaction with γTuRC, the regulator of microtubule nucleation (42). The centrosome is the main site for microtubule nucleation, but most differentiated cells, including epithelial cells, possess nonradial microtubules arranged in noncentrosomal arrays (47). These nonradial microtubules are aligned along the apical-basal axis of the cell; the plus ends are located basally, whereas the minus ends are apical, thus contributing to the structural and functional polarity of epithelia (48). One could consider an enticing hypothesis whereby P. aeruginosa affected the polarity of the cell by recruiting γTuRC to sites of infection at the plasma membrane, in order to create new sites of microtubule nucleation. These new sites would be enriched in microtubule-minus ends, which might interfere with the directional transport of microtubule-dependent cargoes in the cell and thus affect the polarity of the cells (19). One type of cargo could be PI3K, whose recruitment to the apical surface may generate local production of PIP3, creating a basolateral environment at the apical surface and formation of protrusions enriched for PIP3 and actin. Indeed membrane-associated γ-tubulins were found to interact with kinases, among which is PI3K (49). In agreement with this, VgrG2b does not colocalize with the centrosome upon infection of HeLa cells or during intracellular production in HEK293 cells (see Fig. S2 and S5 in the supplemental material), suggesting that VgrG2b may interact with noncentrosomal γTuRCs. An example for noncentrosomal microtubule rearrangement during phagocytosis can be found in nonelicited macrophages, where microtubules are seen extending into the phagosomal cup, in addition to actin cytoskeleton rearrangements (50). Accordingly, colchicine treatment reduces pseudopod formation but also causes a decrease in activated PI3K and Akt (50). Altogether, the findings by Khandani et al. suggest that microtubules regulate the recruitment and localized activity of PI3K and Akt during pseudopod formation. Upon host kinase activation by contact with P. aeruginosa, VgrG2b might then promote microtubule nucleation at the membrane through interaction with γTuRCs that would lead to pseudopod formation for engulfment of the bacterium. One would expect to find noncentrosomal microtubule remodeling during P. aeruginosa internalization in epithelial cells, which we have been unable to observe yet. In concordance with this hypothesis, Salmonella enterica serovar Typhimurium invades fibroblasts by a route differing from the entry into epithelial cells (51). Uptake of S. enterica by fibroblasts requires microtubules and PI3K activation in a type III secretion system (T3SS)-independent way, whereas microtubules and PI3K are dispensable during Salmonella T3SS-based invasion of epithelial cells and macrophages.

Figure S5 

Noncentrosomal localization of VgrG2b upon infection of HeLa cells with the WT PAO1 strain (A) or during intracellular production of VgrG2bHA-Strep in HEK293 cells (B). VgrG2b was labeled with anti-VgrG2b (1/500) (A) or anti-HA (1/1,000) (B) antisera, γ-tubulins 1 and 2 were labeled with anti-GCP1-2 antisera (1/1,000), and proteins were detected with secondary anti-IgG antibodies conjugated to Cy3 and Cy5, respectively. VgrG2b is labeled in green, and γ-tubulins 1 and 2 are labeled in red. Download Figure S5, PDF file, 0.1 MB.
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This raises the question regarding the nature of the C-terminal effector domain of VgrG2b and the phylogenetic distribution of VgrG2b-like proteins. VgrG2b is conserved in all P. aeruginosa strains present in the Pseudomonas genome database. Moreover, a sequence similarity search indicated a strong association of VgrG2b with the Pseudomonas genus. To identify VgrG2b homologues that possess the C-terminal extension, we developed an in silico 3-step strategy (see Text S1 and Fig. S6 in the supplemental material). The phylogenetic tree clearly separated two groups, the VgrG2b of Pseudomonas and those of Burkholderia species, even if other few species are represented, like Acinetobacter (see Fig. S6B and C). Interestingly, all of these proteins contained the DUF2345 domain (PF10106), whereas this criterion has not been included in the search (see Text S1 and Fig. S6A and B), suggesting that this domain is a hallmark of VgrG2b homologues. Unexpectedly, the putative zinc protease domain predicted in VgrG2b (32) was not present in B. cepacia complex (BCC) homologues and seems thus restricted to Pseudomonas (see Fig. S6A and B). Interestingly P. aeruginosa and BCC are both recognized as respiratory pathogens in cystic fibrosis patients. Bacteria residing in the airway lumens proliferate in spite of host attempts to clear them through macrophage and epithelial cell uptake, neutrophil killing, and mucociliary clearance. Some BCC members (B. cenocepacia and B. dolosa) were shown to invade in a microtubule-dependent way the epithelial cells (8). Both pathogens may thus invade respiratory cells via a VgrG2b-mediated entry pathway, thus avoiding rapid elimination by macrophage phagocytosis or mucociliary clearance. Whether this leads to progression of infection is yet to be determined.

Text S1 

Experimental procedures. Download Text S1, DOCX file, 0.03 MB.
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Figure S6 

VgrG2b-like proteins’ phylogeny. (A) Conserved features of VgG2b-like proteins. A sequence similarity search restricted to the last 259 residues of VgrG2b (residues 761 to 1019) retrieved 580 proteins (step 1). Then, a position-specific model made from the multiple alignments of all matches (hidden Markov model) revealed several conserved motifs among these proteins (step 2). The VgrG2b organization is presented with a color code for the domains: PF05954, orange; PF04717, purple; PF13296, pink; the putative Zn protease domain, yellow. Shown is WebLogo sequence alignment of VgrG2b homologues identified by the in silico screen. The VgrG2b domains are underlined with the same color code. (B and C) Maximum likelihood rooted phylogenetic tree of VgrG2b homologues. To construct a phylogenetic tree, the list of homologues has been condensed by keeping proteins harboring a VgrG domain (PF05954, PF04717, and PFG13296) and the VgrG2b C-terminal extension (step 3). The phylogenetic tree was calculated using RAxML version 7.7.9. The scale bar represents the number of estimated changes per position for a unit of branch length. Download Figure S6, PDF file, 0.7 MB.
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In the future, we will further address how P. aeruginosa subverts the microtubule network to invade nonphagocytic cells. The main questions that remain to be answered are what are the effects of the bacteria on cell polarity and recruitment of the microtubule motors and particularly whether this uptake leads to microtubule remodeling.

MATERIALS AND METHODS

Bacterial strains, plasmids, and antiserum production and growth conditions.All P. aeruginosa and E. coli strains used in this study are described in Table S1 in the supplemental material. See the experimental procedures in Text S1 in the supplemental material for the specific growth conditions, cloning procedures, and antisera used in Western blotting.

Table S1 

Strains, plasmids, and oligonucleotides used in this study. Table S1, DOCX file, 0.02 MB.
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HeLa cell invasion assays.To enumerate bacteria internalized by HeLa cells through the H2-T6SS machinery, gentamicin protection assays were performed as described in reference 10 and modified in reference 20 with P. aeruginosa strains grown at the transition between the exponential and stationary phases. HeLa cells (ATCC, Manassas, VA) were cultured to 70% confluence on the day of the experiment in 24-well plates in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). The cells were washed twice with sterile phosphate-buffered saline (PBS), infected with a P. aeruginosa strain, and grown in the late exponential phase at a multiplicity of infection (MOI) of 10 in MEM with FBS at a final volume of 1 ml. Synchronous infection conditions and an enhanced bacterium-host cell interaction were achieved by a 5-min centrifugation step at 1,000 × g. After 3 h of infection at 37°C in 5% CO2, the cells were washed twice with sterile PBS and incubated with MEM supplemented with 300 µg·ml−1 gentamicin (Sigma) for 1 h to kill extracellular bacteria. Afterward, the cells were washed three times with room temperature PBS and lysed at room temperature with 1% Triton X-100 (Sigma). Lysed cells were collected in Eppendorf tubes, and serial dilutions in sterile LB were plated onto Pseudomonas isolation agar plates. Colony-forming units were enumerated and reported as internalized bacteria.

To study the requirement of the microtubules or actin during bacterial internalization, colchicine (Sigma), nocodazole (Sigma), and cytochalasin D (Sigma) were added at various concentrations 30 min (colchicine) or 1 h (nocodazole and cytochalasin D) before infection with P. aeruginosa.

Immunofluorescence staining and image analysis.HeLa cells were plated onto the coverslip in DMEM complemented with 10% FBS medium at 150,000 cells/cm2 and infected with P. aeruginosa, grown at the transition between the exponential and stationary phases, at an MOI of 10 for 3 h. After being rinsed with PBS three times, coverslips were washed and fixed with either 4% paraformaldehyde (PFA)–PBS for 10 min at room temperature or with methanol 100% at −20°C for 15 min. After three washes with PBS, the cells were permeabilized with PBS–0.1% Saponin for 30 min. The coverslips were blocked with 10% bovine serum albumin (BSA)-PBS for 1 h followed by incubations with primary (anti-V5 and anti-VgrG2b, 1/500) and secondary antibodies for 1 h each in 10% BSA–PBS at room temperature. Secondary antibodies were stained with the fluorescence-coupled Cy3- or Cy5-conjugated goat anti-rabbit (A11008) and Cy3- or Cy5-conjugated goat anti-mouse (A11005) antibodies (both from Invitrogen). After another three washes with PBS, coverslips were mounted on glass slides using ProLong Gold (Invitrogen) and left in the dark overnight before the microscopy study. Samples were examined with a confocal microscope (Olympus IX81) using a ×60 oil objective (UPLSAPO; numerical aperture, 1.35). Images were acquired using FLUOVIEW Viewer software.

Affinity purification.Full details of the construction and cell culture of isogenic stable cell lines exhibiting tetracycline (Tet)-inducible expression of recombinant vgrG2a, vgrG2b, and gfp genes can be found in Text S1 in the supplemental Material.

VgrG2aHA-Strep, VgrG2bHA-Strep, and GFPHA-Strep HEK293 Flp-In cell pellets were lysed by incubation in freshly prepared buffer (50 mM HEPES [pH 8.0], 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 1.5 mM Na3VO4, 1.0 mM phenylmethylsulfonyl fluoride [PMSF], and protease inhibitor cocktail) on ice for 20 min. Each lysate was centrifuged at 14,000 × g for 30 min, and the supernatant containing the protein extract was collected. The protein concentration was determined by the Bradford assay using BSA as a standard. Two-step affinity purifications were performed as previously described (52) and detailed in the supplemental material.

Reversed-phase LC-MS.The protein eluates from the two-step affinity purifications were reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. A complete protocol for LC-MS separation and data analysis is presented in the supplemental material.

Coimmunoprecipitation.HEK293 Flp-In T-REx cells carrying the genes for vgrG2aHA-Strep or vgrG2bHA-Strep were cultured in 10-cm petri dishes in Dulbecco's modified Eagle's medium (DMEM) according to the manufacturer's instructions (Invitrogen). The expression of recombinant vgrG2a or vgrG2b was induced by adding doxycycline to the medium to 1 µg/ml for 16 h. Subsequently, cells were scraped off the culture dish and centrifuged, and after two rinses with PBS, cells were lysed with 75 µl/dish of 50 mM HEPES, 100 mM KCl, 1 mM EDTA, 1 mM MgCl2, 1% NP-40, 1 mM β-mercaptoethanol, and a cocktail of protease inhibitors (Roche). Six microliters of ascites fluid containing monoclonal antibody against the HA tag was added (clone HA-7; Sigma-Aldrich), and following incubation for 30 min on ice, immunoprecipitation was performed by adding 50 µl of protein G coupled to magnetic beads (Dynabeads, Invitrogen). Following several rinses, bound protein was eluted from the beads with 40 µl of Laemmli gel electrophoresis buffer containing 2% SDS. Eluted proteins, as well as cell lysates before and after immunoprecipitation, were analyzed by Western blotting. Western blots were probed with mouse-anti-HA (clone HA-7; Sigma-Aldrich), and rabbit antibodies against γ-tubulin or GCP4, respectively (53).

Statistical analyses.Unpaired Student's t tests and the χ2 test were performed using Excel software (Microsoft). On the figures, asterisks indicate statistical significance.

ACKNOWLEDGMENTS

We thank V. Baylot and members of R.V.'s team for constant support, B. Ize and S. Salcedo for careful reading of the manuscript, P. Hubert for training and advice on confocal microscopy, E. Cascales for providing HA-tagged antibodies, and J. van Helden for advice on statistical analysis.

T.G.S. was financed with a Ph.D. fellowship from the French Research Ministry and with a “Teaching and Research” fellowship from AMU, and A.H. was financed with a postdoctoral fellowship from EuroPathoGenomics and Wellcome Trust (WT091939). This work was supported by “Pathomics” ERA-net PATHO grant ANR-08-PATH-004-01. A.F.'s group was financed by MRC grant MR/K001930/1.

FOOTNOTES

    • Received 26 April 2015
    • Accepted 6 May 2015
    • Published 2 June 2015

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

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