Bacterial toxins induce sustained mRNA expression of the silencing transcription factor klf2 via inactivation of RhoA and Rhophilin 1

Abstract

Yersiniae bearing the Yersinia virulence plasmid pYV impact the transcriptome of J774A.1 macrophage-like cells in two distinct ways: (i) by suppressing, in a Yersinia outer protein P (YopP)-dependent manner, the induction of inflammatory response genes and (ii) by mRNA induction of the silencing transcription factor klf2. Here we show that klf2 induction by Yersinia enterocolitica occurs in several cell lines of macrophage and squamous and upper gastrointestinal epithelial origin as well as in bone marrow-derived dendritic cells. Several strains of Pseudomonas aeruginosa and Staphylococcus aureus are equally effective as Y. enterocolitica in inducing klf2 expression. Screening of mutant strains or incubation with recombinant toxins identified the rho-inactivating toxins YopT from Yersinia spp., ExoS from Pseudomonas aeruginosa, EDIN-B from Staphylococcus aureus, and C3bot from Clostridium botulinum as bacterial inducers of klf2 mRNA. klf2 mRNA induction by these toxins does not require de novo protein synthesis. Serum response factor or actin depolymerization does not seem to be involved in regulating klf2 expression in response to bacterial infection. Instead, short hairpin RNA-mediated inactivation of RhoA and its effector rhophilin 1 is sufficient to induce long-term klf2 expression. Thus, bacteria exploit the RhoA-rhophilin signaling cascade to mediate sustained expression of the immunosuppressive transcription factor klf2.

FIG. 1.

klf2 mRNA induction in J774A.1 cells by different gram-positive and gram-negative bacteria (detailed in Table 2). The Y. enterocolitica-elicited klf2 level was scaled to an arbitrary value of 10. Levels of klf2 mRNA were recorded relative to the HPRT housekeeping control. Error bars represent 1 standard deviation from the mean. Mock, uninfected cells. Horizontal brackets or asterisks indicate statistically significant (t test; P ≤ 0.05) differences in klf2 mRNA expression levels. (A) klf2 induction by different clinical isolates of Y. pseudotuberculosis. (B) klf2 induction by a collection of phylogenetically diverse bacteria.

FIG. 2.

Identification of bacterial klf2-inducing proteins. (A) klf2 mRNA expression in J774A.1 cells after infection with different Y. enterocolitica mutant strains (detailed in Table 2). The graphical display is as described in the legend to Fig. 1. *, statistically significant (t test; P ≤ 0.05) induction of klf2 mRNA compared to infection with strain WA-C. (B) klf2 mRNA levels in J774A.1 cells after infection with different P. aeruginosa wild-type and mutant strains (see Table 2). The graphical display is as described in the legend to Fig. 1. Horizontal brackets indicate statistically significant (t test; P ≤ 0.05) differences in klf2 mRNA expression levels. (C) klf2 induction by recombinant EDIN-B or C. botulinum C3 toxin in J774A.1 cells. The graphical display is as described in the legend to Fig. 1. sycE, irrelevant protein used as negative control.

FIG. 3.

klf2 mRNA levels induced by wild-type Y. enterocolitica and YopT mutants in cell lines derived from different human or mouse tissues. The graphical display is as described in the legend to Fig. 1. Note the logarithmic scale. *, statistically significant (t test; P ≤ 0.05) induction of klf2 mRNA compared to infection of the respective cell line with strain WA-C. DC, mouse bone marrow-derived DCs.

FIG. 4.

Effect of shRNA-mediated knockdown of Rho GTPases on klf2 expression levels. (A) rhoA, rhoB, and rhoC mRNA expression levels in rhoA shRNA-transduced cells. (B) rhoA, rhoB, and rhoC mRNA expression levels in rhoB shRNA-transduced cells. (C) rhoA, rhoB, and rhoC mRNA expression levels in rhoC shRNA-transduced cells. (D) Effect of Rho GTPase knockdown on klf2 mRNA expression level.

FIG. 5.

Impact of Rho effector proteins on klf2 expression level. (A) klf2 mRNA levels in J774A.1 cells treated with the ROCK inhibitor H1152 for the indicated times (O/N, overnight) at the indicated concentrations. Similar results were obtained with the inhibitor H1077. (B) Impact of indicated Rho effector protein knockdown by shRNA on klf2 expression level.

FIG. 6.

Proposed model of klf2 regulation in the context of bacterial infection. Immediately after bacterial contact, host cells induce klf2 by an as yet uncharacterized signaling cascade; however, MAPK or NF-κB signaling may be involved. Two hours after infection, signaling via RhoA and rhophilin 1 suppresses klf2 expression. Bacteria mediate long-term expression of klf2 by suppressing this inhibitory action of RhoA via Rho-inactivating enzymes.