Shigella flexneri Regulation of ARF6 Activation during Bacterial Entry via an IpgD-Mediated Positive Feedback Loop

Anna Cristina Garza-Mayers; Kelly A Miller; Brian C Russo; Dipal V Nagda; Marcia B Goldberg

doi:10.1128/mBio.02584-14

. 2015 Mar 3;6(2):e02584-14. doi: 10.1128/mBio.02584-14

Shigella flexneri Regulation of ARF6 Activation during Bacterial Entry via an IpgD-Mediated Positive Feedback Loop

Anna Cristina Garza-Mayers ^a, Kelly A Miller ^b, Brian C Russo ^b, Dipal V Nagda ^b,^*, Marcia B Goldberg ^a,^b,^✉

Editor: Jeff F Miller^c

PMCID: PMC4358011 PMID: 25736891

ABSTRACT

Entry into cells is critical for virulence of the human bacterial pathogens Shigella spp. Shigella spp. induce membrane ruffle formation and macropinocytic uptake, but the events instigating this process are incompletely understood. The host small GTPase ADP-ribosylation factor 6 (ARF6) functions in membrane trafficking at the plasma membrane and activates membrane ruffle formation. We demonstrate that ARF6 is required for efficient Shigella flexneri entry, is activated by S. flexneri dependent on the phosphatase activity of the type III secreted effector IpgD, and depends on cytohesin guanine nucleotide exchange factors (GEFs) for recruitment to entry sites. The cytohesin GEF ARF nucleotide binding site opener (ARNO) is recruited to these sites, also dependent on IpgD phosphatase activity. ARNO recruitment is independent of ARF6, indicating that, in addition to the described recruitment of ARNO by ARF6, ARNO is recruited upstream of ARF6. Our data provide evidence that ARF6, IpgD, phosphoinositide species, and ARNO constitute a previously undescribed positive feedback loop that amplifies ARF6 activation at bacterial entry sites, thereby promoting efficient S. flexneri uptake.

IMPORTANCE

Shigella spp. cause diarrhea and dysentery by infection of epithelial cells in the human colon. Critical to disease is the ability of Shigella to enter into cells, yet the mechanisms involved in entry are incompletely understood. We demonstrate that the small GTPase ADP-ribosylation factor 6 (ARF6) is required for efficient cellular entry of Shigella flexneri and that activation of ARF6 depends on the phosphatase activity of the Shigella protein IpgD, which is introduced into cells via the bacterial type III secretion system. We further show that IpgD phosphatase activity is required for recruitment of the ARF6 guanine nucleotide exchange factor (GEF) ARF nucleotide binding site opener (ARNO) to bacterial entry sites and that ARNO lies upstream of ARF6 activation. These relationships define a positive feedback loop that contributes to activation of ARF6 at S. flexneri entry sites and leads to local amplification of signals that promote bacterial entry.

INTRODUCTION

Shigella spp. are Gram-negative bacterial pathogens that cause diarrheal disease by invading and spreading through the colonic epithelium. Shigella entry into intestinal epithelial cells is critical to the disease process (1, 2) and requires a type III secretion system (T3SS) and secreted effectors (2, 3). Upon contact with nonphagocytic cells, delivery of T3SS effector proteins causes rearrangements of the actin cytoskeleton, which induce plasma membrane ruffling that leads to macropinocytic uptake of the pathogen (4).

Several bacterial and host proteins have been shown to be involved in Shigella entry. Disruption of the activity of individual proteins leads to defects but not to complete abrogation of entry, indicating that entry pathways are partially redundant. Shigella IpaA promotes entry by binding vinculin, but an ipaA mutant still invades at low frequency (5). Actin rearrangements promoted by the small Rho GTPases Cdc42 and Rac1 contribute to entry, yet expression of dominant negative forms of either decreases entry by only ~70% (6). Activation of the Rac1 guanine exchange factor (GEF) DOCK180/ELMO by Shigella IpgB1 contributes to entry, yet an ipgB1 mutant invades at 35% of the efficiency of the wild type (WT) (7, 8).

GTPases are molecular switches that transduce signals to regulate functions as diverse as gene expression, vesicular trafficking, and cytoskeletal rearrangement (9) and are manipulated by several bacterial pathogens (10). GTPases of the ADP-ribosylation factor (ARF) family act at the plasma membrane and at endosomes to regulate membrane trafficking, phosphoinositide and lipid metabolism, and actin remodeling (11, 12). Distinct from other ARF GTPases in amino acid sequence and intracellular localization, GTP-bound ARF6 is localized to the plasma membrane and on endosomes, where it activates phosphatidylinositol 4-phosphate 5-kinase (PIP5K), generating phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] (13 –15). In motile cells, ARF6 also promotes actin remodeling via activation of Rac1, dependent on the GEFs ARF nucleotide binding site opener (ARNO) and DOCK180/ELMO complex (16 –19).

The localization of ARF6 at the plasma membrane and its role in membrane trafficking and actin remodeling during endocytosis place it in an ideal position to participate in pathogen uptake and endosome remodeling (18 –22). The mechanism of ARF6 recruitment and activation has not been described for any pathogen.

We report a role for ARF6 in a previously undescribed positive feedback loop that amplifies ARF6 activation to promote Shigella flexneri entry. We find that ARF6 is activated during S. flexneri entry and is required for efficient entry. We show that the ARF GEF ARNO lies upstream of ARF6 in this pathway and that the activity of the T3SS effector IpgD, an inositol 4-phosphatase (23 –25), is required for recruitment of both ARF6 and ARNO to bacterial entry sites. These findings provide new insights into the mechanism of GTPase recruitment and activation during bacterial entry.

RESULTS

ARF6 is required for efficient entry of S. flexneri.

To test whether ARF6 contributes to S. flexneri infection, we quantified the efficiency of infection in HeLa cells transfected with wild-type (WT) hemagglutinin (HA)-tagged ARF6 or ARF6 N122I, a dominant negative mutant unable to bind guanine nucleotides (21). At 2 h of infection, the number of intracellular bacteria in cells expressing ARF6 N122I was 30% less than in cells expressing WT ARF6 (7.2 ± 0.1 versus 5.1 ± 0.2 intracellular bacteria per transfected cell, P = 0.001) (Fig. 1A), demonstrating that entry and/or early intracellular replication of S. flexneri is less efficient in the presence of dominant negative ARF6. To test whether ARF6 per se is required for efficient infection, we examined infection in mouse embryonic fibroblasts (MEFs) that harbor a stable lentiviral short hairpin RNA (shRNA) construct targeting Arf6 mRNA (ARF6^K/D) (26), such that levels of ARF6 are reduced by >90% compared to control cells (ARF6⁺) that harbor a nontargeting lentiviral insertion (see Fig. S1A in the supplemental material). Viable intracellular S. flexneri levels in ARF6^K/D MEFs were 2-fold decreased compared to ARF6⁺ MEFs at 1 and 2 h of infection (at 1 h, 1,327 ± 311 CFU versus 651 ± 147 CFU, P = 0.03; at 2 h, 11,027 ± 1,430 CFU versus 5,188 ± 193 CFU, P = 0.03) (Fig. 1B). Thus, in both HeLa cells and MEFs, in the absence of ARF6, entry and/or early intracellular replication of S. flexneri is less efficient.

FIG 1 — ARF6 is required for efficient entry of *S. flexneri*. (A) Efficiency of infection of HeLa cells transfected with ARF6-HA or dominant negative ARF6 N122I-HA. Number of intracellular bacteria per cell at 2 h of infection, by differential staining. (B) *S. flexneri* recovered after infection (1 or 2 h) of ARF6^K/D or ARF6⁺ MEFs, by gentamicin protection assay. (C and D, left graph) Early infection (40 min) of ARF6^K/D or ARF6⁺ MEFs, with differential staining of extracellular versus intracellular bacteria. Extracellular bacteria stained with antibody to LPS (green) and all bacteria identified by DAPI (DNA, blue). Phalloidin staining of polymerized actin (red). Images are representative. Arrows, intracellular bacteria. Arrowheads, extracellular bacteria. Bar, 10 µm. (D, right graph) Rescue of *S. flexneri* entry by transient transfection of ARF6^K/D MEFs with ARF6-HA. Efficiency of entry into ARF6-HA-transfected cells was for the subset of cells that expressed ARF6-HA. Mean ± standard error of the mean of at least 3 independent experiments.

S. flexneri entry into cells occurs as early as 15 min after bacterial contact with the host cell (27, 28). To test whether ARF6 is required for S. flexneri entry per se, we quantified the percentage of intracellular bacteria 40 min after initial contact with cells, before significant replication of intracellular bacteria would have occurred, differentiating extracellular bacteria from those that were intracellular by differential labeling. Extracellular bacteria were identified by labeling with S. flexneri lipopolysaccharide (LPS) prior to cell permeabilization, whereas all bacteria were identified by staining DNA with 4′,6-diamidino-2-phenylindole (DAPI). At 40 min of infection, the number of cells infected by S. flexneri was decreased by 75% in ARF6^K/D MEFs compared to ARF6⁺ MEFs (32% ± 8.6% versus 8% ± 0.6% of cells were infected, P = 0.03) (Fig. 1C and D, left graph). When transiently transfected with an HA-tagged WT ARF6 construct, ARF6^K/D MEFs exhibited partial restoration of ARF6 protein levels (see Fig. S1A in the supplemental material). The relatively minor rescue in levels was likely due to the combination of only a portion of the cells having been transfected and the susceptibility of the ARF6-HA RNA to the shRNA expressed in the ARF6^K/D cells. Nevertheless, transient transfection of this construct resulted in rescue of bacterial entry to that in ARF6⁺ MEFs (Fig. 1D, right graph), suggesting that relatively low levels of ARF6 are sufficient to promote entry and demonstrating that efficient S. flexneri entry is dependent on ARF6.

Virulent S. flexneri activates ARF6 during early infection.

Like most small GTPases, in resting cells, ARF6 is predominantly in the GDP-bound inactive form. The requirement for ARF6 for efficient S. flexneri entry suggested that ARF6 might be activated during this process. To quantify ARF6 activation, GTP-bound ARF6 was precipitated from lysates of infected cells using beads conjugated to the ARF binding domain of Golgi complex-associated, gamma adaptin ear-containing ARF binding protein 3 (GGA3), which specifically binds GTP-bound ARF6 (29). WT S. flexneri induced activation of ARF6 as early as 20 min after contact with HeLa cells, whereas a strain that is noninvasive by virtue of lacking the virulence plasmid did not activate ARF6 at either time assessed (Fig. 2A), indicating that S. flexneri activates ARF6 soon after cell contact and in a manner that depends on virulence plasmid-encoded factors.

FIG 2 — Virulent *S. flexneri* activates ARF6 early during infection. (A) ARF6 activation determined by pulldown of GTP-bound ARF6 from HeLa cells 20 or 40 min after initial contact of WT or noninvasive *S. flexneri* (top panel) with cells, versus total ARF6 in each lysate (bottom panel). GTPγS or GDP added to lysates of uninfected cells and uninfected cells alone (uninfect) are positive and negative controls. Ratio of ARF6-GTP to total ARF6 for blots shown, by densitometry. Blots are representative. (B and C) ARF6-HA recruitment to AFA-I-expressing WT or noninvasive *S. flexneri* in HeLa cells infected (40 min), fixed, and stained with antibody to HA (green), DAPI (blue), and phalloidin (red). ND, not detectable. Mean ± standard error of the mean for 3 independent experiments. Images are representative. Arrows, ARF6 recruitment to entering bacteria. Arrowheads, bacteria without ARF6 recruitment. Bar, 10 µm.

Exchange of bound GDP for GTP activates ARF6 and recruits it from a cytosolic pool to the plasma membrane (30 –32), suggesting that ARF6 activation during S. flexneri entry might be associated with its recruitment to entry sites. WT S. flexneri recruited HA-tagged ARF6 to bacteria at the plasma membrane early in infection (40 min), but the noninvasive strain did not (85% versus <1% of cells displayed ARF6 recruitment to bacteria, P = 0.03) (Fig. 2B and C). In this experiment, to ensure that bacterial contact with the plasma membrane occurred independently of type III secretion, we used strains that adhered tightly as a result of heterologous production of the uropathogenic Escherichia coli afimbrial adhesin (AFA-I); as a result, every cell displayed adherent bacteria. S. flexneri similarly recruited ARF6-HA in MEFs (18% versus <1% of cells with bacteria associated showed ARF6 recruitment to bacteria, P = 0.001) (see Fig. S2 in the supplemental material); here, only a subset of cells displayed adherent bacteria, as AFA-I was not used because its receptor is absent in murine cells. These results show that ARF6 activation by S. flexneri is associated with its recruitment to bacterial entry sites and is dependent not simply on contact with the plasma membrane but rather on virulence plasmid-encoded factors.

The phosphoinositide phosphatase activity of Shigella T3SS effector IpgD is required for efficient ARF6 activation at entry sites.

We tested whether any of several effector proteins secreted into cells by the S. flexneri virulence plasmid-encoded type III secretion system (T3SS) were required for activation of ARF6. We postulated that the involved effector was likely to either possess GTPase exchange factor (GEF) activity or recruit an ARF GEF to entry sites. We therefore focused on the effectors IpgB1, IpgB2, IpgD, and IpaJ. IpgB1 and IpgB2 possess GEF activity; IpgB1 principally activates the host GTPases Rac1 and Cdc42, and IpgB2 principally activates RhoA, yet the two effectors are homologous with some overlap in function (8, 33, 34). IpgD is an inositol 4-phosphatase with no known homology to effectors with GEF activity but, like IpgB1, is translocated early upon bacterial contact with host cells and participates in actin remodeling at entry sites (7, 23, 24). Moreover, the substrate of IpgD, PI(4,5)P₂, is concentrated at the plasma membrane (35). IpaJ demyristoylates ARF1 (36), an ARF GTPase that is 66% identical to ARF6 (11).

To test whether any of these effectors is required for S. flexneri-induced ARF6 activation, we examined the ability of strains lacking each individually to activate ARF6 during entry. Whereas the other mutant strains examined activated ARF6 at levels comparable to the WT strain, the ΔipgD mutant was defective in ARF6 activation (Fig. 3A). We then tested whether IpgD was required for ARF6 recruitment to bacterial entry sites by specifically assessing the efficiency of ARF6 recruitment to sites where bacteria were adherent to cells and/or within a membrane ruffle, reasoning that these constituted the bulk of the bacterial population potentially undergoing cellular entry. Compared to WT, the ΔipgD mutant was defective in ARF6 recruitment (57% versus 38% of cells with bacteria associated showed ARF6 recruitment, P = 0.04) (Fig. 3B, left graph). The ARF6 recruitment defect was rescued by ipgD expressed in trans, but not by expression of ipgD C438S, a catalytically dead mutant with no phosphatase activity (24) (54% and 7% of cells with bacteria associated showed ARF6 recruitment, respectively, P = 0.003) (Fig. 3B, right graph, and C). These results demonstrate that IpgD phosphatase activity is critical for ARF6 activation and for ARF6 recruitment to bacterial entry sites.

The ΔipgD mutant was defective in entry (17% of cells infected by ΔipgD mutant versus 31% of cells infected by WT, P = 0.002) (Fig. 3D, left graph), and the entry defect was rescued by WT IpgD but not by IpgD C438S (68% versus 21% of cells infected, respectively; P = 0.0006) (Fig. 3D, right graph). Thus, IpgD phosphatase activity is required both for activation of ARF6 and for efficient entry. The ΔipgD mutant was defective in the formation of plasma membrane ruffles at entry sites (25% versus 12% of cells displayed bacteria in a ruffle; P = 0.001) (Fig. 3E, left graph). IpgD but not IpgD C438S rescued the ruffling defect (46% and 4% of cells displayed bacteria in a ruffle, respectively; P = 0.0016) (Fig. 3E, right graph). Both the ΔipgD mutant complemented with WT IpgD and that complemented with IpgD C438S produced IpgD at higher levels than the WT strain (see Fig. S3 in the supplemental material), likely due to plasmid copy number. In the presence of overexpression of WT IpgD, entry and ruffle formation were more efficient, whereas overexpression of IpgD C438S was associated with an apparent dominant negative effect on entry and ruffle formation, further confirming a role for IpgD and IpgD phosphatase activity in S. flexneri entry.

Efficient ARF6 recruitment to entering S. flexneri requires cytohesin GEFs.

GEFs activate small GTPases such as ARF6 by stimulating GDP dissociation, which allows GTP binding. In the absence of homology that might suggest intrinsic GEF activity, a mechanism by which IpgD might promote ARF6 activation is by recruiting and/or activating an ARF6 GEF at S. flexneri entry sites. Known GEFs of ARF6 include BRAG1, GEP100/BRAG2, EFA6, and the cytohesin GEFs, cytohesin-1, ARNO (cytohesin-2), and GRP1 (cytohesin-3) (37 –41). We tested whether cytohesin GEFs are required for S. flexneri entry and for ARF6 recruitment to entering bacteria by treating cells with SecinH3, a small-molecule cytohesin family-specific inhibitor (42) that inhibits all cytohesin family GEFs. Cytohesin-1 is highly expressed in natural killer T lymphocytes (43), whereas ARNO and GRP1 have roles in epithelial cells (17, 44). SecinH3 inhibited S. flexneri entry (30% versus 11% of cells were infected; P = 0.04) (Fig. 4A) and ruffle formation (29% versus 10% of cells displayed bacteria in a ruffle; P = 0.02) (Fig. 4B), indicating that one or more cytohesin GEFs contribute to entry. SecinH3 inhibited S. flexneri entry not only in the presence of ARF6 (29% versus 14% of ARF6⁺ MEFs were infected, P = 0.03) but also in its absence (16% versus 8% of ARF6^K/D MEFs were infected, P = 0.02) (data not shown); since ARNO can activate ARF1 downstream of ARF6 (18, 19), we speculate that this SecinH3-induced decrease in infection of ARF6^K/D MEFs may be due to inhibition of ARNO-dependent activation of ARF1.

FIG 4 — Efficient ARF6 recruitment to entering *S. flexneri* requires cytohesin GEFs. Recruitment of ARF6-HA to HeLa cells infected with *S. flexneri* for 40 min in the presence of SecinH3 or dimethyl sulfoxide (DMSO). (A) Percentage of cells infected. (B) Percentage of cells with ruffles. (C) Percentage of cells with bacteria associated showing ARF6 recruitment to bacteria. Mean ± standard error of the mean of at least 3 independent experiments. (D) Representative images with labeling with antibody to HA (green), DAPI (blue), and phalloidin (red). Arrows, bacteria with ARF6 recruitment. Arrowheads, bacteria without ARF6 recruitment. Bar, 10 µm.

To determine whether cytohesin GEFs were functioning upstream of ARF6 in S. flexneri entry, we tested whether ARF6 recruitment depended on cytohesin activity. In the presence of SecinH3, ARF6 recruitment to potential entry sites was decreased (44% versus 15% of cells with bacteria associated showed ARF6 recruitment to bacteria; P = 0.001) (Fig. 4C and D), demonstrating that SecinH3 blocked efficient ARF6 activation by S. flexneri. Thus, cytohesin GEF activity is critical to efficient ARF6 activation by entering S. flexneri and to efficient bacterial entry, consistent with the inhibitory effect of SecinH3 on entry being due at least in part to inhibition of ARF6 activation.

Interaction of IpgB1 with ELMO, a component of the Rac1 GEF DOCK180/ELMO complex, promotes bacterial uptake (7). ARNO is required for activation of DOCK180/ELMO (17). Whether ARNO activation of DOCK180/ELMO depends on ARF6 is unclear. These observations, in conjunction with the observed effects of SecinH3 on S. flexneri activation of ARF6 and entry, led us to postulate that ARNO might participate in activation of ARF6. Myc-tagged ARNO was recruited to S. flexneri entry sites. Experimental optimization suggested that membrane ruffling and ARF6 recruitment both peak within 30 to 40 min after initial bacterial contact with HeLa cells, whereas ARNO recruitment peaks approximately 10 min earlier (data not shown). ARNO recruitment in HeLa cells depended on the presence of the virulence plasmid, even for bacteria that expressed the AFA-I adhesin (55% of cells for WT versus <1% of cells for noninvasive strain displayed ARNO recruitment to bacteria; P = 0.001) (Fig. 5A and B).

FIG 5 — ARNO is recruited to entering *S. flexneri* independent of ARF6. (A and B) Recruitment of Myc-ARNO 3G to AFA-I-expressing WT or noninvasive *S. flexneri* in HeLa cells, infected for 30 min. Representative images labeled with antibody to Myc (green), DAPI (blue), and phalloidin (red). (C and D) Recruitment of Myc-ARNO 2G or Myc-ARNO 3G to *S. flexneri* entering ARF6^K/D or ARF6⁺ MEFs, infected for 25 min. Labeling as in panel B. Arrows, entering bacteria with ARNO recruitment. Arrowheads, bacteria without ARNO recruitment. ND, not detectable. NS, not significant. Mean ± standard error of the mean of at least 3 independent experiments. Bars, 10 µm.

Since ARNO recruitment apparently preceded ARF6 recruitment, we tested whether ARNO recruitment might be independent of ARF6 by examining its recruitment in ARF6⁺ and ARF6^K/D MEFs. ARNO exists as two splice variants—ARNO 2G and ARNO 3G—that differ by the addition of a single glycine residue in the phosphoinositide-binding pocket of the ARNO pleckstrin homology (PH) domain (45). Because the two variants display distinct binding preferences for phosphoinositide species at the membrane (45, 46), we separately examined the recruitment of each. Each ARNO variant was independent of ARF6 for recruitment to entering bacteria (42% each of cells with bacteria associated showed ARNO 2G recruitment to bacteria in ARF6⁺ and ARF6^K/D MEFs, respectively; P = 1.0; for ARNO 3G, 42% versus 35%, respectively; P = 0.3) (Fig. 5C and D), indicating that ARNO is recruited upstream of ARF6, which positions it to potentially contribute to activation of ARF6 at sites of S. flexneri entry. These findings are notably different from what has been observed during Salmonella infection, where ARNO recruitment to entry sites is defective in the absence of ARF6 (19); the reasons for these organism-based differences are at present unclear.

IpgD and IpgD phosphatase activity are required for ARNO recruitment to entry sites and S. flexneri entry.

IpgD-induced PI(5)P accumulation activates PI 3-kinase (PI3K), leading to generation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (47). The PH domains of ARF GEFs bind phosphoinositides with various affinities; these interactions recruit GEFs to the plasma membrane (37, 48 –50). We considered that recruitment of ARNO to S. flexneri entry sites might be enhanced by IpgD-mediated generation of phosphoinositide species that bind ARNO with high affinity. IpgD was required for recruitment of ARNO to entry sites (cells with bacteria associated showing ARNO recruitment to bacteria, 59% for WT versus 20% for ΔipgD mutant, P = 0.001) (Fig. 6A, left graph). IpgD but not IpgD C438S rescued ARNO recruitment by the ΔipgD mutant (44% and 5% of cells with bacteria associated showed ARNO recruitment to bacteria, respectively) (Fig. 6A, right graph, and B). In the absence of IpgD, ARNO recruitment was more dramatically reduced than ARF6 recruitment (Fig. 6A, left graph, versus 3B), likely due to a contribution of other SecinH3-sensitive ARF6 GEFs to ARF6 recruitment, since knockdown of ARNO alone did not lead to significant decreases in ARF6 recruitment (see Fig. S4 in the supplemental material). As above (Fig. 3D and E), the phosphatase activity of IpgD was required for both efficient entry and membrane ruffling at entry sites (see Fig. S5 in the supplemental material). Moreover, PI(3,4,5)P₃ was generated at S. flexneri entry sites, as assessed by recruitment of a green fluorescent protein (GFP)-tagged pleckstrin homology domain from Bruton’s tyrosine kinase (BTK-PH-GFP), which specifically binds PI(3,4,5)P₃ (51), and efficient generation of PI(3,4,5)P₃ was dependent on IpgD (ΔipgD mutant, 18%, versus WT, 37%, for cells displaying BTK-PH-GFP recruitment to entry sites, P = 0.04) (Fig. 6C and D). Thus, the phosphatase activity of IpgD is critical for both generation of PI(3,4,5)P₃ at entry sites and ARNO recruitment, and IpgD and SecinH3-sensitive GEFs are each required for ARF6-mediated S. flexneri entry.

FIG 6 — IpgD phosphatase activity is required for ARNO recruitment to entry sites, and PI 3-kinase contributes to ARNO 2G but not ARNO 3G recruitment. (A and B) WT, Δ*ipgD*, Δ*ipgD* pIpgD, and Δ*ipgD* pIpgD C438S *S. flexneri* infection for 30 min of HeLa cells transfected with Myc-ARNO 3G. (A) Percentage of cells with ARNO recruitment to entering bacteria. (B) Representative images, labeled with antibody to Myc (green), DAPI (blue), and phalloidin (red). (C and D) Recruitment of PI(3,4,5)P₃ to bacterial entry sites upon WT or Δ*ipgD* infection for 40 min of HeLa cells transfected with BTK-PH-GFP, which specifically binds PI(3,4,5)P₃. (C) Percentage of cells with BTK-PH-GFP recruitment to entering bacteria. (D) Representative images, labeled with DAPI (blue), and phalloidin (red), and with GFP signal. (E and F) Inhibition of recruitment of ARNO 2G, but not ARNO 3G, upon treatment with LY294002 upon WT infection of ARF6^K/D MEFs for 25 min. (E) Percentage of cells with ARNO recruitment to entering bacteria. (F) Representative images, labeled as in panel B. Arrows, bacteria with ARNO recruitment. Arrowheads, bacteria without ARNO recruitment. Bars, 10 µm. NS, not significant. Mean ± standard error of the mean of at least 3 independent experiments. DMSO, dimethyl sulfoxide; LY, LY294002.

Recruitment to entry sites of ARNO 2G but not ARNO 3G is defective upon inhibition of PI3K.

These data raised the possibility that IpgD-dependent changes in phosphoinositide composition, specifically PI3K-dependent generation of PI(3,4,5)P₃, contribute to recruitment of ARNO to bacterial entry sites. We tested whether inhibition of PI3K by addition of the chemical inhibitor LY294002 to cells during bacterial entry altered recruitment of ARNO. Recruitment of ARNO 2G, which preferentially binds PI(3,4,5)P₃, was defective in the presence of LY294002 (29% versus 50% of cells with bacteria attached displaying ARNO recruitment, P = 0.04) (Fig. 6E, left graph, and F), whereas recruitment of ARNO 3G, which binds PI(4,5)P₂ with high affinity (45, 46), was unaffected (30% versus 36%, P = 0.4) (Fig. 6E, right graph). Treatment of cells with LY294002 was associated with defective phosphorylation of Akt (see Fig. S6 in the supplemental material), confirming that the conditions used inhibited PI 3-kinase activity. Thus, IpgD-dependent generation of PI(3,4,5)P₃ during entry of S. flexneri enhances recruitment of the 2G variant of the ARF6 GEF ARNO.

DISCUSSION

Our findings, combined with published data, support the presence of a positive feedback loop that promotes ARF6 activation during S. flexneri entry into cells. We demonstrate that activation of ARF6 is required for efficient entry of S. flexneri into both epithelial cells and MEFs, that ARF6 activation depends on phosphatase activity of the T3SS effector IpgD, and that the cytohesin ARF6 GEF ARNO is recruited upstream of ARF6 where it contributes to ARF6 activation (Fig. 7). ARF6 activation is known to drive Rac1-dependent actin remodeling, which promotes membrane ruffle formation and bacterial entry (16, 17). Thus, the amplification of signals immediately beneath sites of bacterial contact with the plasma membrane that result from this positive feedback loop would be expected to promote rapid local changes that facilitate bacterial entry.

FIG 7 — Model of IpgD-dependent positive feedback loop that amplifies ARF6 activation during entry of *S. flexneri*. Extracellular *S. flexneri* recruits ARNO to the plasma membrane in part via IpgD-induced local accumulation of PI(3,4,5)P₃. ARNO-dependent activation of ARF6 induces PIP5K-mediated changes in phosphoinositide composition at the membrane that promote additional ARNO recruitment, mediating further activation of ARF6. Solid arrows, relationships supported by data presented here or previously published. Dashed arrows, postulated relationships.

In a positive feedback loop, amplification allows the output signal to reach a critical threshold at which the system switches from one state to another, traditionally basal to active (52). Positive feedback loops have been reported for Rab (53, 54) and ARF (55, 56) GTPase signaling. ARNO recruitment to the plasma membrane is by two distinct mechanisms: binding to phosphoinositides and phosphatidylserine and binding to ARF6 (57, 58). In an ARF6 feedback loop, GTP-bound ARF6 stimulates ARNO, and ARNO activates ARF1 and is then stimulated by GTP-bound ARF1 (55). Cohen et al. found that GTP-bound ARF6 binds the ARNO PH domain, leading to ARNO recruitment to the plasma membrane and activation of ARF1 (57). Cascades of GEFs and GTPases can thus constitute positive feedback loops that feed back onto themselves.

The positive feedback loop that our data support is distinct from described GEF cascades in that IpgD phosphatase activity generates signals that contribute to GEF recruitment. These signals include the generation of membrane-anchored phosphoinositide species that bind ARNO. Since PI(4,5)P₂, the substrate for IpgD phosphatase activity, is generated by ARF6-mediated activation of PIP5K, by our model, IpgD activity is fueled by its ability to generate phosphoinositides that enhance ARNO recruitment. Moreover, because inactive ARF6 does not bind the ARNO PH domain (57), IpgD-generated phosphoinositides may function as the seed for ARNO recruitment and subsequent ARF6 recruitment and activation. This model, supported by the data presented here, predicts that IpgD activity expands the existing GEF-ARF cascade in a manner that further amplifies ARF6 activation.

Our findings establish a substantial expansion of the model of ARF GTPase participation in entry, previously described for Salmonella enterica serovar Typhimurium by Humphreys et al. (18, 19). Data from the prior studies support a model in which ARF6 recruits ARNO, ARNO in turn recruits ARF1, and ARF1 promotes actin polymerization via the WAVE regulatory complex (18, 19). In our work here, in addition to showing that entry by S. flexneri parallels that of S. Typhimurium with respect to participation of ARF GTPases, we characterize for the first time a mechanism of activation of ARF6 in this process and demonstrate the presence of the previously undescribed positive feedback loop that likely amplifies ARF6 activation. Moreover, our results identify an important difference from the entry of S. Typhimurium, wherein ARNO recruitment is dependent on ARF6 (19), in that during entry of S. flexneri, ARNO recruitment is independent of and occurs upstream of ARF6. Although the reasons are currently unclear, these findings may highlight the existence of important functional divergence between Shigella and Salmonella.

An important insight that emerges from our data together with published data (18, 19) is that during bacterial entry, ARNO participates in the activation of both ARF6 and ARF1, which lies downstream of ARF6. Thus, ARF GEFs such as ARNO (but perhaps not restricted to ARNO) may participate promiscuously at multiple steps during bacterial infection. ARNO recruitment was absolutely dependent on IpgD. The effector IpgB1 recruits the Rac1 GEF DOCK180/ELMO to entry sites (7). DOCK180/ELMO is activated by ARF6 via ARF1 and dependent on ARNO GEF activity (17, 19).

PI(4,5)P₂ is relatively abundant in both resting and stimulated cells, whereas PI(3,4,5)P₃ is detectable only following stimulation (59). IpgD phosphatase activity leads to the recruitment of PI(3,4,5)P₃ (Fig. 6C and D). PI(4,5)P₂ and PI(3,4,5)P₃ bind PH domains of ARF GEFs. The high-affinity interaction of membrane-anchored PI(3,4,5)P₃ with the ARNO 2G variant is sufficient to recruit it to the plasma membrane, whereas the low-affinity interaction of PI(3,4,5)P₃ with the ARNO 3G variant is not sufficient (45, 60, 61). In brain tissue, the 3G variant of ARNO is most abundant (46); the relative abundance of the two variants in other tissues is unknown.

Recruitment of ARNO to S. flexneri entry sites is dependent on IpgD phosphatase activity (Fig. 6A to C) and independent of ARF6 (Fig. 5). The reduction in recruitment of the 2G variant of ARNO upon chemical inhibition of PI 3-kinase (Fig. 6E and F) is consistent with a model in which transient PI(3,4,5)P₃ accumulation, induced by IpgD-dependent activation of PI 3-kinase, leads to efficient recruitment of ARNO 2G via its high-affinity interaction with PI(3,4,5)P₃. However, recruitment of ARNO 3G is independent of PI(3,4,5)P₃ accumulation (Fig. 6D) and thus must be mediated by other factors that accumulate at entry sites in response to IpgD phosphatase activity.

Our data demonstrate for the first time that IpgD is required for efficient S. flexneri entry. Of note, although statistically significant, the defect in entry of the ipgD mutant is relatively minor (Fig. 3D). That the relatively small defect is meaningful is supported by the enhanced entry observed upon overexpression of IpgD (Fig. 3D). The presence of only a minor defect is also consistent with several lines of evidence that point to Shigella entry resulting from multiple partially redundant mechanisms mediated by various T3SS effectors (5, 7, 8). The relatively small decrease observed, along with differences in strain backgrounds, likely explains why the role of IpgD in entry has been unappreciated in other experimental systems (62, 63).

IpgD phosphatase activity and the phosphoinositides that it generates have previously been implicated in S. flexneri activation of Akt (47), regulation of vesicular trafficking (64), recruitment of Rab-11-containing vesicles to bacterial uptake vacuoles (63), inhibition of chemokine-induced migration of T cells (65), inhibition of ATP release through connexin hemichannels (66), and activation of the GEF Tiam1 in Rac1-mediated actin rearrangements (67). Our data now add to the list a role in entry via activation of ARF6. The multiple effects of IpgD during S. flexneri infection highlight the central role that phosphoinositides play in intracellular signaling and the extent to which S. flexneri has evolved mechanisms to manipulate these signaling pathways.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Strains and plasmids are listed in Table 1. All S. flexneri strains are isogenic to serotype 2a wild-type strain 2457T (68). S. flexneri bacteria were grown at 37°C in tryptic soy broth from individual Congo red-positive colonies. Antibiotics were ampicillin (Amp), 100 µg/ml; chloramphenicol (Cm), 25 µg/ml; and kanamycin (Km), 50 µg/ml. To generate pIpgD and pIpgD C438S, the coding sequences of ipgD, its promoter, and its chaperone ipgE were cloned into pACYC184 with site-directed mutagenesis of pIpgD (QuikChange mutagenesis kit; Stratagene).

TABLE 1 .

Bacterial strains and plasmids used in this study

Strain or plasmid	Genotype or description	Reference or source
Bacterial strains
Wild type	Serotype 2a S. flexneri strain 2457t	68
Noninvasive	BS103 (2457T cured of its virulence plasmid)	71
Wild type with adhesin	2457T pIL22, Amp^r	Lab stock
Noninvasive with adhesin	BS103 pIL22, Amp^r	Lab stock
ΔipgD mutant	2457T ipgD::FRT-Km^r-FRT^a	Gift of C. Lesser
ΔipgD pIpgD mutant	2457T ipgD::FRT-Km^r-FRT pACYC184 ipgD-ipgE	This study
ΔipgD pIpgD C438S mutant	2457T ipgD::FRT-Km^r-FRT pACYC184 ipgD C438S-ipgE	This study
ΔipgB1 mutant	2457T ipgB1::FRT	Lab stock
ΔipgB1 ΔipgB2 mutant	2457T ipgB1::FRT ipgB2::FRT	Lab stock
ΔipaJ mutant	2457T ipaJ::FRT	Lab stock
Plasmids
pACYC184	Cm^r	Lab stock
pIpgD	pACYC184 ipgD-ipgE, Cm^r	This study
pIpgD C438S	pACYC184 ipgD C438S-ipgE, Cm^r	This study
pIL22	pBR322 encoding Escherichia coli afimbrial adhesin AFA-I, Amp^r	72
ARF6-HA	pcDNA HA-tagged ARF6, Amp^r	21
ARF6 N122I-HA	pcDNA HA-tagged ARF6 defective for nucleotide binding, Amp^r	21
BTK-PH-GFP	pEGFP-N1-BTK-PH-GFP, Km^r	51
Myc-ARNO 3G	pcDNA3 Myc-tagged ARNO, 3G variant, Amp^r	29
Myc-ARNO 2G	pcDNA3 Myc-tagged ARNO, 2G variant, Amp^r	Gift of L. Santy

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^{^a}

FRT, FLP recombination target.

Cell culture.

HeLa cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco), and ARF6^K/D and ARF6⁺ mouse embryonic fibroblasts (MEFs) (gift of Wim Annaert [26]) were cultured in DMEM–nutrient mixture F-12, each with 10% fetal bovine serum. Monolayers were transfected 24 h prior to infection with 1.5 µg DNA and Fugene 6 transfection reagent (Promega). Knockdown of ARNO was performed using the ARNO-specific small interfering RNAs (siRNAs) Hs_PSCD2_2 SI00061292, Hs_PSCD2_3 SI00061299, Hs_PSCD2_6 SI03050894, and Hs_PSCD2_7 SI03072629 or AllStars negative-control siRNA SI03650318 (Qiagen) and HiPerFect transfection reagent (Qiagen).

Infection of cells.

Bacteria (optical density at 600 nm [OD₆₀₀], 0.3 to 0.4) were added to cell monolayers at a multiplicity of infection (MOI) of 200 to 300:1 (bacteria to cell) or, for strains containing pIL22, at an MOI of 10:1. Cells were centrifuged at 700 × g for 10 min and then incubated at 37°C for a specified additional period of time. SecinH3 (2.5 μM; Calbiochem) or LY294002 (50 µM; Cell Signaling; catalog no. 9901S) was added immediately prior to addition of bacteria. Infected monolayers were washed, fixed in 3.7% paraformaldehyde for 15 to 20 min, and permeabilized with 1% Triton X-100. Labeling was performed using antibodies to S. flexneri 2a lipopolysaccharide (LPS; Gibco) or HA (Covance MMS-101P) or with Myc (Clontech 631206), phalloidin (Invitrogen), or DAPI (100 μM; Invitrogen).

Gentamicin protection assay.

Cells seeded at 3 × 10⁵ per well of a 6-well dish were infected at an MOI of 0.02:1 (69); incubated at 37°C for 5 or 50 min; washed three times; incubated for an additional 45 or 60 min, respectively, in DMEM containing 50 µg/ml gentamicin to kill extracellular bacteria; and lysed with 0.5 to 1% Triton X-100. Bacteria in cell lysates and in the initial inoculum were determined by plating.

Protein production and secretion.

Synthesis and secretion of IpgD were assayed essentially as described previously (70). Proteins were resolved by SDS-PAGE and probed with IpgD (gift of A. Phalipon [24]) or DnaK (StressGen) antibody.

Pulldown assay.

GTP-bound ARF6 in infected monolayers was measured per the manufacturer’s instructions (Cytoskeleton, Inc.). Briefly, cells were infected at an MOI of 300:1 (as described above), followed by incubation for an additional 10 or 30 min, lysis on ice, and harvesting and snap-freezing of the lysate. Two hundred fifty micrograms of each lysate was incubated with beads covalently conjugated to the ARF6 binding domain of GGA3 for 1 h at 4°C. Beads were washed and recovered. Bound ARF6 was resolved by SDS-PAGE and analyzed using ARF6 antibody (gift of J. Donaldson). Other antibodies used were phosphor-Akt S473 and pan-Akt (Cell Signaling) and ARNO (Abcam ab56510).

Microscopy and data analysis.

Microscopy was performed on a Nikon Eclipse TE300 or TE2000 microscope with Chroma Technology filters. For all sets of experiments, three or more independent experiments were performed. Samples were blinded. At least 10 infected cells were analyzed for each condition. A bacterium was counted as entering the cell if it was found associated with a membrane ruffle, determined by actin staining and phase microscopy, and as potentially undergoing bacterial entry if it was adherent and/or within a membrane ruffle. A cell was counted as infected if at least one bacterium was entering or present within the cell. The significance of differences between two-way comparisons was determined by paired t test. The significance of differences among multiple sets of data was determined by one-way analysis of variance (ANOVA) followed by a post hoc Tukey comparison.

SUPPLEMENTAL MATERIAL

Figure S1

Arf6 knockdown. Whole-cell lysates of ARF6^K/D and ARF6⁺ MEFs, transfected with ARF6-HA where indicated. Immunoblotting with ARF6 or β-actin antibodies. Download

Figure S1, TIF file, 0.6 MB^{(596.9KB, tif)}

Figure S2

S. flexneri recruits ARF6 to entry sites in MEFs. (A) Recruitment of ARF6-HA to WT or noninvasive S. flexneri in ARF6⁺ MEFs infected for 25 min. ND, not detectable. Mean ± standard error of the mean of 3 independent experiments. (B) Representative images, labeled with antibody to HA (green), DAPI (blue), and phalloidin (red). Arrows, entering bacteria with ARF6 recruitment. Arrowheads, bacteria without ARF6 recruitment. Bar, 10 µm. Download

Figure S2, TIF file, 2.7 MB^{(2.6MB, tif)}

Figure S3

IpgD production in complemented ΔipgD S. flexneri. Congo red-induced secretion of S. flexneri strains. Pellets and precipitated supernatants immunoblotted with antibodies to IpgD or DnaK. Download

Figure S3, TIF file, 0.2 MB^{(224.1KB, tif)}

Figure S4

Recruitment of ARF6-HA to entering bacteria following siRNA depletion of ARNO in HeLa cells. (A) Depletion of ARNO by siRNA. Immunoblotting with ARNO or β-actin antibodies. (B) Percentage of cells with bacteria associated showing ARF6-HA recruitment to bacteria. Mean ± standard error of the mean of at least 3 independent experiments. NS, not significant. Download

Figure S4, TIF file, 1.4 MB^{(1.4MB, tif)}

Figure S5

In cells transfected with ARNO constructs, IpgD phosphatase activity is required for efficient S. flexneri entry and ruffle formation. (A) Percentage of cells infected. (B) Percentage of cells with ruffles. Mean ± standard error of the mean of at least 3 independent experiments. Download

Figure S5, TIF file, 0.3 MB^{(264.9KB, tif)}

Figure S6

LY294002 treatment that inhibits recruitment of ARNO 2G is associated with inhibition of Akt activation. Levels of phospho-Akt S473 and total Akt under conditions used for data presented in Fig. 6. Western blotting with β-actin as loading control. Download

Figure S6, TIF file, 0.2 MB^{(177.4KB, tif)}

ACKNOWLEDGMENTS

We acknowledge C. R. Yi and S. Y. Lee for bacterial strain construction and B. B. Herrera for technical support. We thank W. Annaert, J. G. Donaldson, L. C. Santy, R. R. Isberg, A. Phalipon, H. Ploegh, and C. F. Lesser for providing reagents; V. W. Hsu for helpful advice; and J. R. Mayers for critical reading of the manuscript.

This work was supported by Public Health Service grant R01AI081724 (to M.B.G.), trainee support on T32GM007753 (to A.C.G.-M.), and T32AI007061 (to K.A.M. and B.C.R.) from the National Institutes of Health.

Footnotes

Citation Garza-Mayers AC, Miller KA, Russo BC, Nagda DV, Goldberg MB. 2015. Shigella flexneri regulation of ARF6 activation during bacterial entry via an IpgD-mediated positive feedback loop. mBio 6(2):e2584-14. doi:10.1128/mBio.02584-14.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Arf6 knockdown. Whole-cell lysates of ARF6^K/D and ARF6⁺ MEFs, transfected with ARF6-HA where indicated. Immunoblotting with ARF6 or β-actin antibodies. Download

Figure S1, TIF file, 0.6 MB^{(596.9KB, tif)}

Figure S2

Figure S2, TIF file, 2.7 MB^{(2.6MB, tif)}

Figure S3

Figure S3, TIF file, 0.2 MB^{(224.1KB, tif)}

Figure S4

Figure S4, TIF file, 1.4 MB^{(1.4MB, tif)}

Figure S5

Figure S5, TIF file, 0.3 MB^{(264.9KB, tif)}

Figure S6

Figure S6, TIF file, 0.2 MB^{(177.4KB, tif)}

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PERMALINK

Shigella flexneri Regulation of ARF6 Activation during Bacterial Entry via an IpgD-Mediated Positive Feedback Loop

Anna Cristina Garza-Mayers

Kelly A Miller

Brian C Russo

Dipal V Nagda

Marcia B Goldberg

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

RESULTS

ARF6 is required for efficient entry of S. flexneri.

FIG 1 .

Virulent S. flexneri activates ARF6 during early infection.

FIG 2 .

The phosphoinositide phosphatase activity of Shigella T3SS effector IpgD is required for efficient ARF6 activation at entry sites.

FIG 3 .

Efficient ARF6 recruitment to entering S. flexneri requires cytohesin GEFs.

FIG 4 .

FIG 5 .

IpgD and IpgD phosphatase activity are required for ARNO recruitment to entry sites and S. flexneri entry.

FIG 6 .

Recruitment to entry sites of ARNO 2G but not ARNO 3G is defective upon inhibition of PI3K.

DISCUSSION

FIG 7 .

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

TABLE 1 .

Cell culture.

Infection of cells.

Gentamicin protection assay.

Protein production and secretion.

Pulldown assay.

Microscopy and data analysis.

SUPPLEMENTAL MATERIAL

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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