Role for Myosin II in Regulating Positioning of Salmonella-Containing Vacuoles and Intracellular Replication

HeLa and RAW 264.7 cells were obtained from the ATCC and maintained in Dulbecco's modified Eagle's medium (HyClone) supplemented with 10% fetal bovine serum (Wisent) at 37°C with 5% CO₂ and without antibiotics. Cells were used between passages 5 and 30.

The serovar Typhimurium strains used in this study were CS401 (wild type [WT]) (49), CS800 (ΔssaT) (31), MBO87 (ΔsseJ) (51), MBO107 (ΔsseJ; complemented with a plasmid carrying sseJ) (51), and MBO106 (ΔsseJ; complemented with a plasmid encoding a catalytically inactive mutant of SseJ [with mutations S151A, D247N, and H384N]) (51), which have all been described previously. MBO207 (ΔpipB2) was generated in the CS401 background. WT serovar Typhimurium SL1344 (35) and isogenic mutants including M202 (ΔsopE ΔsopE2 mutant) (70), ΔsopB (68), a ΔsopB mutant complemented with sopB (68), and a ΔsopB mutant complemented with a plasmid carrying the catalytically inactive (C462S) mutant of sopB (68) have all been described previously. Serovar Typhimurium SL1344/psifA-2HA (12) was used for the SifA colocalization experiments.

Bacteria were grown in Luria-Bertani (LB) broth supplemented with streptomycin, carbenicillin, kanamycin (all at 50 μg/ml), or chloramphenicol (30 μg/ml) as required.

Plasmids used were pegfp-N1 (Clontech); pmrlc2-WT and pmrlc2-AA (19) (generously provided by G. Egea, University of Barcelona); psopBΔGFP (42); prhoA-Q63L, pCdc42-Q61L, and prhoG-Q61L (33) (generously provided by W. D. Heo and T. Meyer, Stanford University); and pDN-ROCK (44), pCAT-ROCK (2), and pMLC-DD (36) (generously provided by A. Kupas, University Health Network, Toronto, Ontario, Canada). Plasmids were transfected into cells 16 h before infections/immunostaining using Fugene 6 (Roche) or Gene Juice (Promega) transfection reagents according to manufacturers’ instructions.

Salmonella infections were performed as previously described (69). Briefly, bacteria were grown for 16 h at 37°C with shaking and then subcultured (1:33) for 3 h in LB broth. Late-log-phase bacteria were used at a multiplicity of infection of approximately 300:1 to infect cells with a brief (10-min) exposure to bacteria. Drugs (from Sigma; see below) were added at 10 min p.i. and cells were fixed at 2 h p.i. or drugs were added at 2 h p.i. and cells were fixed at 8 h p.i. in 20 μM blebbistatin, 0.5 μg/ml cytochalasin D, 2 μg/ml nocodazole in 1% dimethyl sulfoxide (DMSO), 5 μM Y-27632 in 1% DMSO, or 2 nM calyculin A in 1% DMSO.

To knock down expression of myosin IIA and ROCK I and ROCK II, small inhibitory RNAs (siRNAs) (Dharmacon) were used. HeLa cells were seeded into 24-well culture plates at 2.5 × 10⁴ cells per well. The following day, cells were transfected using Oligofectamine (Invitrogen) with either control siRNA (pool of four nontargeting siRNAs [siCONTROL, D-001206-13-05; Dharmacon]) or siRNA directed against myosin IIA (siGENOME SMARTpool, M-007668-00; Dharmacon) or both ROCK I (5′-GAG GCT CAA GAC ATG CTT A-3′) and ROCK II (5′-GGC ATC GCA GAA GGT TTA T-3′) (Dharmacon). A concentration of 50 nM of total siRNA was used in each knockdown. Medium was changed 24 h after transfection, and HeLa cells were infected with serovar Typhimurium 48 h after transfection.

Intracellular SCV positions were determined by measuring the distances from lysosome-associated membrane protein-1-positive (LAMP-1⁺) SCVs to the nearest edge of the nucleus (labeled by DAPI [4′,6′-diamidino-2-phenylindole]). Images of infected cells were acquired by epifluorescence microscopy. Measurements were determined using Openlab 3.1.7 software (Improvision). The distances from ≥100 LAMP-1⁺ SCVs to the nucleus were measured for each time point. Average distances ± standard errors (SE) for three separate experiments were determined. Average cell surface areas were also determined by tracing cell outlines (>30 cells per time point or drug treatment for each of three separate experiments) imaged by epifluorescence or differential interference contrast microscopy and determining surface area by using Openlab software.

Cells were fixed for 10 min at 37°C in phosphate-buffered saline (PBS), pH 7.2, containing 2.5% paraformaldehyde. Fixed cells were washed with PBS and then permeabilized/blocked in PBS-10% normal goat serum containing 0.2% saponin. For phospho-myosin II light chain (PP-MLC) staining, cells were washed for 10 min with PBS-100 mM glycine prior to permeabilization. Samples were incubated separately with primary and secondary antibodies diluted in the PBS-10% normal goat serum containing 0.2% saponin for 1 h each and then washed three times with PBS. Coverslips were mounted onto glass slides by using antifade mounting reagent (DakoCytomation) and were analyzed by using a Leica DMIRE2 epifluorescence microscope.

For confocal microscopy, cells were immunostained as described above and then analyzed on a spinning-disk confocal microscope. Confocal sections of 0.25 μm were acquired by using a Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu back-thinned electron multiplying charge-coupled-device camera and spinning-disk confocal scan head. Volocity 4 (Improvision) software was used to assemble confocal z sections into flattened projections and for movie construction. Image assembly was done using Adobe PhotoShop and Adobe Illustrator software.

Mouse anti-LAMP-1 H4A3 monoclonal antibody (used at a dilution of 1:50) developed by T. August was obtained from the Developmental Studies Hybridoma Lab under the auspices of the NICHD, National Institutes of Health Sciences, and maintained by the University of Iowa, Iowa City, IA. Bacteria were detected by using either anti-Salmonella group B rabbit polyclonal antibodies from Difco at 1:100 or a monoclonal anti-Salmonella antibody (BioDesign International) at 1:100. Actin was detected by using Alexa Fluor 568-conjugated phalloidin (Molecular Probes) at 1:50, and rabbit polyclonal myosin IIA heavy chain antibodies from Covance were used at 1:200. Transfected cells were detected using either a monoclonal antibody to the myc epitope tag (1:50) (Covance), a polyclonal anti-green fluorescent protein (GFP) antibody (1:200) (Clontech), or a monoclonal anti-GFP antibody (1:200) (Invitrogen). Phospho-myosin II was detected with the rabbit Ser 18/Thr19 phospho-myosin II antibody from Cell Signaling (1:50 for immunofluorescence, 1:1,000 for Western blotting). Hemagglutinin (HA) was detected using the monoclonal HA.11 (Covance) at 1:100 for 4 h. A rabbit polyclonal kinesin antibody against the kinesin heavy chain was used at 1:500 (PCP42; a gift from Ron Vale, University of California). The following secondary antibodies were used at 1:200: Alexa Fluor 568-conjugated goat anti-rabbit immunoglobulin G (IgG), Alexa Fluor 350-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-rat IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG, and Alexa Fluor 568-conjugated goat anti-mouse IgG (all from Molecular Probes). For some experiments, mouse anti-LAMP-1 H4A3 was conjugated to Alexa Fluor 568 by using the Zenon Alexa Fluor 568 mouse IgG labeling kit (Molecular Probes). To detect myosin II during Western blotting with siRNA-treated cell extracts, rabbit anti-nonmuscle myosin heavy chain (BT-561) (Biomedical Technologies Inc.), which detects both myosin IIA and IIB isoforms, was used at 1:1,000. To detect ROCK II, rabbit anti-ROKα/ROCK II (clone A9W4) (Upstate) was used at 1:1,000.

HeLa cells were seeded into 24-well culture plates at 2.5 × 10⁴ cells per well. The following day, cells were transfected with Lipofectamine 2000 (Invitrogen) with indicated constructs. Cells were washed with cold PBS (without magnesium and without calcium), and cell lysates were collected 17 h following transfection.

For all experiments, average values and standard deviations (SD)/SE for three experiments were determined, and ≥100 bacteria/SCVs/Sifs/cells were counted for each treatment/sample within an experiment. Depending on the nature of the experiment, statistical analyses were performed using either a two-tailed unpaired t test or a one-way analysis of variance (ANOVA) (Prism 4 software). P values of <0.05 were considered significant.

Disruption of the host cell actin cytoskeleton during serovar Typhimurium infection causes destabilization of the SCV, resulting in bacterial escape into the cytosol (46). To investigate the potential effect of actin motors on SCV stability, we used blebbistatin to inhibit the nonmuscle myosin II in HeLa cells. In control cells, ∼78% of the WT bacteria were present in SCVs at 8 h p.i., as determined by colocalization with the SCV marker LAMP-1 (Fig. 1A). In contrast, blebbistatin-treated cells had only 58% of bacteria in SCVs, suggesting that myosin II regulates SCV stability. We saw, besides decreased SCV stability, an increase in the number of intracellular bacteria in blebbistatin-treated cells by 10 h p.i. (Fig. 1B), consistent with the observation that the cytosol of HeLa cells permits serovar Typhimurium replication (8, 13). We noted that myosin II inhibition with blebbistatin did not affect the efficiency of bacterial entry into cells (data not shown). In HeLa cells infected with the isogenic (SPI-2-deficient) ΔssaT mutant (31), myosin II inhibition had no effect on SCV stability; the numbers of bacteria in SCVs were ∼75% in control and drug-treated cells (Fig. 1A). Hence myosin II inhibition leads to destabilization of SCVs in a manner dependent on the SPI-2 T3SS.

Growth of serovar Typhimurium in vivo is correlated with the ability to replicate in macrophages (20), so we tested whether myosin inhibition in macrophages affects SCV stability and/or replication. RAW 264.7 cells were infected for 2 h with WT or ΔssaT serovar Typhimurium, and DMSO or blebbistatin was added to the media for an additional 8 h. In contrast to the case for HeLa cells, blebbistatin treatment of RAW 264.7 cells did not affect SCV stability (Fig. 1C). However, at 10 h p.i., ∼39% of control cells contained >15 bacteria, while the percentage of such blebbistatin-treated cells was ∼15% (Fig. 1D). Decreased bacterial growth without a decrease in SCV stability may occur if myosin inhibition affects macrophage trafficking. Since the macrophage cytosol is cytotoxic for serovar Typhimurium (8), LAMP-1-negative bacteria may also have been degraded by 10 h p.i.

SseJ is a SPI-2 effector with deacylase activity (51) and antagonizes the SCV-stabilizing effect of SifA (60). To see if SseJ plays a role in bacterial escape from SCVs in the absence of myosin II activity, we infected HeLa cells for 8 h with WT, ΔsseJ, or ΔsseJ bacteria complemented with plasmids encoding WT SseJ or a catalytically inactive mutant of SseJ (51) in the presence of DMSO or blebbistatin. In contrast to the SCV stability of the WT strain, SCV stability of the ΔsseJ strain was unaffected by blebbistatin, with ∼75% LAMP-1⁺ bacteria in control and drug-treated HeLa cells (Fig. 1A). Complementation of ΔsseJ bacteria with WT psseJ plasmid restored the destabilizing effect of blebbistatin, unlike the expression of a catalytically inactive SseJ mutant (Fig. 1A). Hence, the deacylase activity of SseJ is required to destabilize the SCV in the absence of myosin II activity.

Recent studies have suggested that, in addition to SCV stability, the movement of the SCV to the juxtanuclear region is dependent on microtubule motors (25, 28). Therefore, we asked whether the actin cytoskeleton also plays a role in this positioning. HeLa cells were infected with WT serovar Typhimurium and then immediately (10 min p.i.) treated with the actin-depolymerizing agent cytochalasin D. At 2 h p.i., cells were fixed and immunostained for bacteria and LAMP-1. To measure SCV positioning, the distances from intracellular bacteria within vacuoles (LAMP-1⁺) to the closest nuclear edge were determined, as performed in other studies (1, 10, 11, 28, 29), and these values were averaged for each experiment (see Materials and Methods). For control (DMSO-treated) cells, we observed that SCVs were 2.7 ± 0.5 μm from the nucleus (Fig. 2A and B). We were concerned that cytochalasin D treatment would affect cell morphology and hence influence the positions of SCVs relative to the nucleus. Therefore, we measured the surface area of the infected cell at the interface with the glass coverslip by using Openlab software (see Materials and Methods). Even though use of cytochalasin D resulted in some shrinkage of HeLa cells (as determined by a drop in surface area [data not shown]), SCVs in these cells were significantly farther from the nucleus than the SCVs in the untreated controls were (Fig. 2B). This suggested that actin is important for SCV positioning.

Next, we treated HeLa cells with the myosin II inhibitor blebbistatin, as described above. After 2 h of treatment, SCVs of the blebbistatin-treated cells were significantly farther from the nucleus than those of the controls were (Fig. 2A and B). Treatment with blebbistatin did not induce significant changes in infected host cell surface areas in comparison to those of untreated, infected controls (Fig. 2C); hence, the effects of the drug on SCV positioning were not due to grossly disrupted cell morphology. Blebbistatin treatment did not affect Golgi apparatus or lysosome distribution, nor did it affect the dynamics of Rab5 (early endosomal), Rab11 (recycling endosome), or LAMP-1 acquisition on the SCV (data not shown), indicating that the effect of blebbistatin on SCV localization was not due to altered maturation of this compartment. SCVs in nocodazole-treated cells were farther from the nucleus than those in controls were, but the difference was not statistically significant (P = 0.13) (Fig. 2B). The average cell surface area of nocodazole-treated cells did not differ significantly from the average cell surface area of DMSO-treated controls (data not shown).

Next, we inhibited myosin II by using a dominant negative myc-tagged MLC construct that cannot be phosphorylated (MRLC2-AA) and is therefore inactive (19). As controls, HeLa cells were transfected with GFP or a myc-tagged WT myosin regulatory light chain construct (MRLC2-WT) (19). Transfected cells were infected for 2 h with WT serovar Typhimurium, fixed, and immunostained, and the distances from SCVs to the nucleus in transfected cells were measured (Fig. 2D and E). Bacteria were on average 3.1 ± 0.02 μm from the nucleus in GFP-transfected cells and 2.8 ± 0.13 μm from the nucleus in MRLC2-WT-transfected cells (Fig. 2E). In contrast, SCVs in MRLC2-AA-transfected cells were significantly farther from the nucleus (5.8 ± 0.5 μm) (Fig. 2E). Measurements of average cell surface areas did not reveal any significant differences between control GFP-transfected cells and those transfected with MRLC2-WT or MRLC2-AA (Fig. 2F). These findings suggest that myosin II plays a major role in regulating movement of the SCV from the host cell periphery to the perinuclear region during early infection.

During late stages of infection (8 to 14 h p.i.), SCVs are maintained at a perinuclear position through actions of SPI-2 T3SS effectors that modulate microtubule motor activity (1, 10, 25, 29, 58). To investigate a role for actin in SCV positioning at later stages of infection, we treated HeLa cells with cytochalasin D at 2 h p.i. (after the bacteria had moved to the perinuclear region) and incubated them for an additional 6 h. Despite some host cell shrinkage in the presence of cytochalasin D, SCVs in these drug-treated cells were still farther from the nuclear edge (7.6 ± 1.1 μm) than were SCVs in control cells (3.3 ± 0.5 μm) (Fig. 3B). Blebbistatin treatment also led to a peripheral dispersion of SCVs at this time point (Fig. 3A and B). The average cell surface area of infected blebbistatin-treated cells did not differ significantly from that of the infected DMSO-treated controls (data not shown). Nocodazole treatment had no effect on SCV positioning (Fig. 3B).

To further confirm the role of myosin II in SCV positioning, we used siRNA to knock down myosin IIA expression in HeLa cells prior to invasion with WT serovar Typhimurium, since myosin IIA is the dominant isoform of myosin II in HeLa cells (77). As shown in Fig. 3C, treatment of cells with siRNA against myosin IIA resulted in an increase in the average distance between SCVs and the nucleus at 8 h p.i. in comparison to the value for the cells treated with control siRNA. There was no significant difference in average cell surface area between infected control siRNA- and myosin IIA siRNA-treated cells (Fig. 3C). Western blotting using antibody directed against both myosins IIA and IIB verified the knockdown of myosin IIA in HeLa cells treated with myosin IIA siRNA, with densitometric analysis indicating a >80% knockdown in comparison to control cells (Fig. 3C). Together, these results demonstrate that myosin II regulates SCV positioning during both early and late stages of infection.

It was not immediately obvious why myosin II inhibition caused centrifugal displacement of SCVs at 8 h p.i. We hypothesized that microtubule motors, such as kinesin, might mediate centrifugal SCV displacement in the absence of myosin II activity and that SPI-2 effectors may promote this. To test this, HeLa cells were infected with WT or isogenic ΔssaT (SPI-2 T3SS-defective) serovar Typhimurium strains for 2 h, then blebbistatin was added to the media, and this was followed by an additional 6-h incubation. As expected, WT bacteria were peripherally displaced from the nucleus in the presence of the drug (Fig. 3D and E). However, ΔssaT bacteria were not affected by blebbistatin treatment and remained perinuclear (Fig. 3E). This suggested that SPI-2 effector(s) would mediate outward displacement of the SCV in the absence of myosin II activity.

In the absence of SifA, PipB2 promotes accumulation of kinesin on the SCV, leading to its centrifugal displacement (29). Hence, we hypothesized that PipB2 mediates centrifugal SCV displacement in the absence of myosin II activity as well. Compared to its isogenic WT strain, the ΔpipB2 strain maintained a juxtanuclear position at 8 h p.i. in the presence of blebbistatin (Fig. 3D and E). These findings show that PipB2 promotes outward movement of the SCV when myosin II activity is inhibited.

SCV positioning is also regulated by SifA, which interacts with the host protein SKIP to uncouple kinesin from the SCV (10). In the absence of sifA, SCVs accumulate kinesin and are situated away from the nucleus (Fig. 4A, third row) (10), in contrast to the juxtanuclear positioning of WT SCVs at 8 h p.i. (Fig. 4A, top row). To determine if myosin inhibition affects SifA and kinesin recruitment, we infected HeLa cells with WT serovar Typhimurium expressing psifA-2HA and treated cells with blebbistatin. Neither SifA colocalization with SCVs (Fig. 3F and G) nor kinesin recruitment to WT SCVs (Fig. 4A, second row, and B) was affected in blebbistatin-treated cells. Therefore, inhibiting myosin function is sufficient to cause centrifugal SCV displacement without increasing kinesin levels on SCVs.

Activation of myosin II is regulated by phosphorylation of MLC by several kinases, including ROCK (3, 75). ROCK also indirectly promotes MLC phosphorylation by inhibiting myosin II phosphatase, thus maintaining myosin II in its active state (78). Since serovar Typhimurium can modulate several members of the Rho family, including Cdc42, Rac, and RhoG (27, 54, 80), we attempted to determine a role for ROCK in myosin activation during serovar Typhimurium infection. HeLa cells were infected and treated with the ROCK inhibitor Y-27632 at 10 min p.i. After 2 h, internalized bacteria in the drug-treated cells were displaced farther from the nucleus than the bacteria in the controls were (Fig. 5A and B). ROCK inhibition also prevented the SCV from localizing to the perinuclear region at 8 h p.i. (Fig. 5B). Treatment of cells with Y-27632 did not grossly affect host cell morphology, as determined by comparing average cell surface areas of drug-treated and control cells (data not shown). Transfection of HeLa cells with a dominant negative Rho-associated kinase construct (DN-ROCK) (44) also caused centrifugal displacement of SCVs, but transfection with a constitutively active ROCK II catalytic-domain construct (CAT-ROCK) (2) or GFP did not (Fig. 5C). Treatment of HeLa cells with siRNA specific to ROCK I and ROCK II also resulted in an increase in the average distance of SCVs from the nucleus at 8 h p.i. (Fig. 5D). Inhibition of ROCK I and II did not affect the average cell surface area of treated cells in comparison to that of cells receiving control siRNA (data not shown). Western blotting verified >85% knockdown of ROCK II in HeLa cells treated with ROCK I and ROCK II siRNA (Fig. 5D). Commercially available antibodies did not detect ROCK I in Western blotting of HeLa cell lysates, possibly due to its low expression in this cell type. Overall, these data show that a known activator of myosin II, namely, ROCK, is required for normal SCV positioning throughout infection.

Our experiments suggested that SCV positioning might be regulated by members of the Rho GTPase family, acting via ROCK activation. The SPI-1 T3SS effectors SopE, SopE2, and SopB promote invasion by activating host cell Rho GTPases. SopE and SopE2 function as guanine nucleotide exchange factors that activate Cdc42 and Rac1 (27, 70), while SopB is a phosphoinositide phosphatase that activates Cdc42 and RhoG by an unknown mechanism(s) (54, 80). SopE/SopE2 are rapidly degraded following bacterial entry (39). Since SopB protein levels persist in the host cell for up to 12 h p.i. (17), we hypothesized that this protein may regulate SCV positioning. To test this idea, we infected HeLa cells with WT or ΔsopB serovar Typhimurium for up to 10 h and measured the distance from SCVs to the nucleus. For a positive control, cells were infected with a ΔsifA mutant, since SifA controls SCV positioning by uncoupling kinesin from the vacuole (10). At 10 h p.i., SCVs containing the ΔsopB and ΔsifA mutants were significantly farther from the nucleus than the WT bacteria were (Fig. 6A and B). Similar results were seen at 2, 4, and 8 h, suggesting that SopB controls SCV positioning throughout infection (data not shown). In contrast, cells infected with a ΔsopE ΔsopE2 double mutant strain showed no SCV positioning defects (data not shown). Complementation of the ΔsopB strain with a plasmid encoding WT SopB restored normal SCV positioning near the nucleus. However, a plasmid encoding SopB with a mutation in the conserved catalytic site (C462S) (43) did not complement the ΔsopB phenotype (Fig. 6B). myc-tagged SopB was localized to SCVs up to 10 h p.i. (Fig. 7A). This suggests that SopB acts through its phosphatase activity to regulate SCV positioning.

Recently, Brawn et al. showed that another SPI-1 effector, SipA, recruits SifA to the SCV (11). To see if SopB acts similarly, we infected cells with a WT strain with psifA-2HA or a ΔsopB strain with psifA-2HA for 8 h and immunostained for bacteria, HA, and LAMP-1. SifA colocalized with both the WT and ΔsopB mutant SCVs (Fig. 7B and C). Since SifA downregulates kinesin on the SCV (10), we also tested whether kinesin recruitment to the SCV was altered in ΔsopB-infected HeLa cells. No kinesin recruitment to either WT or ΔsopB mutant SCVs was seen (Fig. 4A, first and fourth rows, and B). In contrast, up to 35% of the ΔsifA mutant SCVs colocalized with kinesin (Fig. 4A, third row, and B). These data suggest that SopB acts independently of SifA to regulate SCV positioning.

Our studies suggest that SopB, by activating Rho family GTPases, mediates SCV positioning via myosin II activation. We hypothesized that activation of myosin II by pharmacologic or genetic means would complement positioning of the ΔsopB mutant SCV. HeLa cells were infected with ΔsopB bacteria in the presence or absence of calyculin A, a myosin II phosphatase inhibitor that maintains myosin activity (26). In control cells, ΔsopB mutant SCVs were 5.4 ± 0.3 μm from the nucleus at 8 h p.i. However, in the presence of calyculin A, they localized close to the nuclear edge (2.7 ± 0.3 μm) (Fig. 8A and B). Hence, inhibition of myosin II phosphatase activity (i.e., maintaining activation of myosin II) restored normal intracellular positioning of the ΔsopB mutant SCV. Similar results were obtained with ΔsopB bacterium-infected cells by prior transfection with constitutively active mutants of Rho GTPases (RhoA with the Q63L mutation [RhoA-Q63L], Cdc42 with the Q61L mutation [Cdc42-Q61L], and RhoG with the Q61L mutation [RhoG-Q61L]) (Fig. 8C and D). Furthermore, transfection of HeLa cells with a constitutively active MLC (36), CAT-ROCK, or SopB was sufficient to complement ΔsopB mutant positioning (Fig. 8E). The average cell surface areas of the various transfected cells were not significantly different from GFP-transfected controls, except for RhoA-Q63L-transfected cells, which were smaller than the controls (Fig. 8F). Overall, this shows that the capacity for normal SCV positioning can be restored to ΔsopB bacteria by activating myosin II, suggesting that myosin II acts downstream of SopB during serovar Typhimurium infection.

We infected HeLa cells with serovar Typhimurium for 2 h and immunoblotted infected cell extracts with an antibody specific for MLC phosphorylated at its two activation site residues, Ser18/Thr19 (PP-MLC). Infection with WT serovar Typhimurium did not lead to a global increase in PP-MLC in comparison to uninfected cells or cells infected with ΔsopB bacteria (data not shown). The majority of cellular myosin II remained associated with actin stress fibers during infection, and very little was detected on SCVs (Fig. 9A, top row). Therefore, global levels of PP-MLC would not be predicted to change during serovar Typhimurium infection. Interestingly, blebbistatin treatment allowed us to detect a significantly greater association of myosin II with both SCVs and Sifs (Fig. 9A, second and third rows, and B).

As an alternative strategy to determine if SopB is sufficient for myosin II activation (via phosphorylation of MLC), HeLa cells were transfected with SopB, RhoA-Q63L, Cdc42-Q61L, RhoG-Q61L, or GFP and immunostained for PP-MLC. Transfection of SopB resulted in the accumulation of PP-MLC on actin stress fibers, which was not observed for control GFP-transfected cells (Fig. 10A). Cells transfected with RhoA-Q63L showed the most robust PP-MLC staining, while SopB, Cdc42-Q61L, and RhoG-Q61L had intermediate effects (Fig. 10A and B). Cells transfected with SopB also demonstrated an increase in PP-MLC levels (similar to those of calyculin A-treated cells) compared to those of untreated controls and GFP-transfected controls (approximately 90% and 24% higher levels, respectively) according to Western blotting results (Fig. 10C). Overall, these results demonstrate that SopB expression is sufficient to mediate MLC phosphorylation and myosin II activation.