Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein

Abstract

Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.

Keywords: CNF-Y; Cdc42; Cytotoxic Necrotizing Factor-Y; Rac; Rho; Rho GTPases; Toxins; Type III Secretion System; Yersinia enterocolitica.

FIGURE 1.

CNF-Y increases Y. enterocolitica effector translocation into target cells. A, left panel, HeLa cells were treated with GST-CNF-Y or GST for 2 h and subsequently infected with WA-314 for 5–120 min as indicated. Cells were lysed with digitonin, and supernatants were analyzed for YopH and actin by Western blotting (WB) and densitometry of respective protein bands. Bars show the YopH/actin ratio of the densitometric values. Middle panel, HeLa cells were treated with GST-, GST-CNF-Y-, or GST-CNF-1 for 2 h and infected for 1 h with WA-314. Bars represent the mean ± S.D. (error bars) of YopH/actin ratio normalized to control (GST-treated cells) of at least three independent experiments. Asterisks indicate significant differences when compared with control (one-way analysis of variance with Dunnett's test; *, p = 0.01–0.05 and **, p = 0.001–0.01). Right panel, GST-, GST-CNF-Y-, or GST-CNF-1-treated HeLa cells were infected with WA-314 or Y. pseudotuberculosis III (pIB 102) for 1 h. A digitonin lysis assay was conducted, and supernatants were analyzed for YopE and actin by Western blotting. B, GST- or GST-CNF-Y-treated HeLa cells were preloaded with CCF4-AM substrate and then infected with WA-314-pMK-bla (translocating YopE-β-lactamase fusion) and WA-314-pMK-ova (translocating YopE-ovalbumin fusion; negative control). Hydrolysis of CCF4 substrate by translocated β-lactamase was monitored by live cell imaging. Excitation at 409 nm resulted in green fluorescence emission (520 nm) of the intact substrate and in blue fluorescence emission (447 nm) of the cleaved hydrolysis product. Depicted merged images (green + blue fluorescence) were taken from representative movies at the indicated time points (see also supplemental Movie S1). GST-CNF-Y-treated cells show more rapid accumulation of the hydrolysis product. C, live cell data were quantitatively analyzed by calculation of the fluorescence index Q = (P_cells − P_background)/(S_cells − S_background) to correct for background fluorescence and loss of CCF-4 substrate from the cells. Graphs represent the mean ± S.D. (error bars) of three independent experiments, each condition sampled in triplicate. D, HeLa cells were infected with WA-314 or WA-314ΔE for 1 h. Infection was then terminated by adding gentamicin (50 μg/ml) and chloramphenicol (20 μg/ml). After 30 min of incubation, GST or GST-CNF-Y was added for 4 h. Digitonin lysates were prepared directly after infection (1) and after GST (2) or GST-CNF-Y (3) treatment. Supernatants were analyzed for YopH and actin by Western blot. Data are representative of three independent experiments.

FIGURE 2.

Profiles and kinetics of RhoA, Rac1, and Cdc42 activation by CNF-Y and CNF-1 and effect of constitutively active RhoA, Rac1, and Cdc42 constructs on Yop translocation. A, HeLa cells were treated with GST-CNF-Y or GST-CNF-1 as indicated. Active RhoA, Rac1, or Cdc42 was precipitated with GST-Rhotekin (RhoA) or GST-PAK-CRIB (Rac1, Cdc42) and analyzed by Western blotting. The input represents ∼4% of the cell lysate used for the respective pulldown at the indicated time point. Immunoblots of pulldown assays and inputs were investigated with the same antibodies. Data are representative of three independent experiments. B, HeLa cells were transfected with the constitutively active constructs Myc-RhoAL63, Myc-Rac1L61, or Myc-Cdc42L61. After 16 h, cells were infected with WA-314 for 1 h, digitonin-lysed, and analyzed by Western blotting. Bars represent the mean ± S.D. (error bars) of YopH/actin ratio normalized to control (GST-treated cells) of three independent experiments. Asterisks indicate significant differences when compared with control (one-way analysis of variance with Dunnett's test; *, p = 0.01–0.05 and **, p = 0.001–0.01). ns, not significant.

FIGURE 3.

Knockdown or knock-out of Rac1 blocks CNF-Y stimulated Yop translocation. A, HeLa cells were transfected with nontargeting (n. t.), Rac1, or Cdc42 siRNA for 72 h and then treated with GST or GST-CNF-Y for 2 h. Subsequently, cells were infected with WA-314 for 1 h and digitonin-lysed, and translocated YopH was analyzed by Western blotting (WB). Mean YopH/actin ratio and S.D. of three independent experiments are stated. B, Rac1^−/− and Rac1^fl/fl control MEFs were treated for 2 h with GST or GST-CNF-Y and subsequently infected with WA-314-pmk-bla for 3 h. Live cell imaging data were analyzed and quantified as described above. Graphs represent the mean ± S.D. (error bars) of three independent experiments, each condition sampled in triplicate.

FIGURE 4.

RhoA, -B, and -C and downstream targets are dispensable for Y. enterocolitica effector translocation under basal and CNF-stimulated conditions. A, HeLa cells were treated with nontargeting (n. t.) siRNA or with a siRNA pool for 48 h to simultaneously deplete RhoA, -B, and -C, stimulated with GST-CNF-Y or GST-CNF-1 for 2 h, and infected with WA-314 for 1 h. Cells were lysed with digitonin, and supernatants were analyzed for YopH, RhoA, and actin by Western blotting. Data are representative of three independent experiments. B, HeLa cells were treated as above, preloaded with CCF4-AM, and infected with WA-314-pMK-bla for 3 h. Live cell imaging data were analyzed and quantified as described above. Graphs represent the mean ± S.D. (error bars) of three independent experiments, each condition sampled in triplicate. C, left panel, Alexa Fluor 568 phalloidin staining was conducted of nontargeting, RhoA, or RhoA, -B, and -C siRNA-treated HeLa cells stimulated with CNF-Y for 2 h. Abrogation of stress fiber formation was only seen in the RhoA, -B, and -C siRNA-treated cells, verifying the efficient knockdown of RhoA, -B, and -C. Right panel, siRNA knockdown of RhoA, -B, and -C was also confirmed by Western blot. RhoB expression was very low under control conditions, but was found to be up-regulated when RhoA and -C were depleted. In this case, up-regulation was efficiently counteracted by RhoB siRNA. D, HeLa cells were treated with GST or GST-CNF-Y and simultaneously subjected to different concentrations of small inhibitors blebbistatin and Y-27632 or dimethyl sulfoxide (DMSO) alone for 2 h. Evaluation of stress fiber formation was conducted by Alexa Fluor 568 phalloidin staining to determine effectiveness of inhibitors. After infection with WA-314 for 1 h, cells were lysed with digitonin, and supernatants were analyzed for YopH and actin by Western blotting. Data are representative of three independent experiments.

FIGURE 5.

YopE/T/O cannot counteract the CNF-Y-mediated hypertranslocation. A, HeLa cells were treated with GST or GST-CNF-Y for 2 h and subsequently infected with WA-314 and Yop mutants WA-314ΔE, WA-314ΔE/ΔT, and WA-314ΔE/ΔT/ΔO for 1 h. Digitonin lysates were analyzed for YopH and actin by Western blotting (WB). Data are representative of three independent experiments. B, GST- and GST-CNF-Y-treated HeLa cells were infected with WA-314 and WA-314ΔE for 1 h. Digitonin lysates were analyzed for YopH and actin by Western blotting. Data are representative of three independent experiments.