Characterization of Pyrin Dephosphorylation and Inflammasome Activation in Macrophages as Triggered by the Yersinia Effectors YopE and YopT

Abstract

Pathogenic Yersinia species deliver Yop effector proteins through a type III secretion system into host cells. Among these effectors, YopE and YopT are Rho-modifying toxins, which function to modulate host cell physiology and evade immune responses. YopE is a GTPase-activating protein (GAP) while YopT is a protease, and they inhibit RhoA by different modes of action. Modifications to RhoA are sensed by pyrin, which, once activated, assembles a caspase-1 inflammasome, which generates cytokines such as interleukin-1β (IL-1β) and cell death by pyroptosis. In Yersinia-infected macrophages, YopE or YopT triggers inflammasome assembly only in the absence of another effector, YopM, which counteracts pyrin by keeping it inactive. The glucosyltransferase TcdB from Clostridium difficile, a well-studied RhoA-inactivating toxin, triggers activation of murine pyrin by dephosphorylation of Ser205 and Ser241. To determine if YopE or YopT triggers pyrin dephosphorylation, we infected lipopolysaccharide (LPS)-primed murine macrophages with ΔyopMYersinia pseudotuberculosis strains expressing wild-type (wt) or YopE mutant variants or YopT. By immunoblotting pyrin after infection, we observed that wt YopE triggered dephosphorylation of Ser205 and inflammasome activation. Pyrin dephosphorylation was reduced if a YopE variant had a defect in stability or RhoA specificity but not membrane localization. We also observed that wt YopT triggered pyrin dephosphorylation but more slowly than YopE, suggesting that YopE is dominant in this process. Our findings provide evidence that RhoA-modifying toxins trigger activation of pyrin by a conserved dephosphorylation mechanism. In addition, by characterization of YopE and YopT, we show that different features of effectors, such as RhoA specificity, affect the efficiency of pyrin dephosphorylation.

Keywords: Yersinia; inflammasome; macrophages; pyrin.

FIG 1

Analysis of IL-1β release and pyroptosis in BMDMs. LPS-primed BMDMs were left uninfected or infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE for 90 min at an MOI of 30. As a positive control, TcdB was used to intoxicate BMDMs for 90 min at a concentration of 0.2 μg/ml. Supernatants were collected and analyzed by ELISA for released IL-1β (A) or by measurement of LDH release for pyroptosis (B). Data represent average values ± the standard errors of the means from three independent experiments. Significant differences compared to results for BMDMs infected with IP6ΔM harboring the empty vector were determined by one-way ANOVA. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected, infected with Y. pseudotuberculosis strain IP6ΔM harboring an empty vector or IP6ΔM/pYopE or intoxicated with TcdB for 90 min. (A and B) Lysates were processed with nondenaturing NP-40 lysis buffer and separated into soluble and insoluble fractions by centrifugation. (C) Lysates were processed with denaturing RIPA lysis buffer, and the soluble fractions were collected. Samples were subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

FIG 3

Analysis of IL-1β release in BMDMs. LPS-primed BMDMs were left uninfected or infected with Y. pseudotuberculosis IP6ΔM/pYopE or IP6ΔMmJ mutants harboring empty vector, pYopE^R62K, pYopE^L109A, pYopE^3N, or pYopE for 90 min. After infection, supernatants were collected and analyzed by ELISA to detect the release of IL-1β. Data represent average values ± the standard errors of the means from three independent experiments. Significant differences compared to results with IP6ΔMmJ/pYopE-infected BMDMs were determined by one-way ANOVA. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected or infected with the indicated Y. pseudotuberculosis IP6ΔM or IP6ΔMmJ mutants for 90 min at an MOI of 30. After infection, lysates were processed with denaturing RIPA lysis buffer, collected, and subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

FIG 5

Analysis of IL-1β release and pyroptosis in BMDMs. LPS-primed BMDMs were left uninfected or infected with a Y. pseudotuberculosis 32777 ΔyopM, yopE^R144A, yopT^C139A, or yopE^R144A yopT^C139A strain for 90 min. Supernatants were collected and analyzed by ELISA for release of IL-1β (A) or by measurement of LDH release for pyroptosis (B). Data represent average values ± the standard errors of the means from three independent experiments. Significant differences compared to results for the 32777 ΔyopM or yopE^R144A mutant-infected BMDMs were determined by one-way ANOVA. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 6

Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected or infected with the indicated Y. pseudotuberculosis 32777 ΔyopM strain for 90 min. After infection, lysates were processed with denaturing RIPA lysis buffer, collected, and subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.

FIG 7

Analysis of IL-1β release in BMDMs. LPS-primed BMDMs were left uninfected or infected with indicated Y. pseudotuberculosis 32777 ΔyopM strains for 90 min or 180 min. Supernatants were collected and analyzed by ELISA to detect the release of IL-1β. Data represent average values ± the standard errors of the means from three independent experiments. Significant differences comparing the results of the same strains or different strains at the two time points were determined by two-way ANOVA. ns, not significant; **, P < 0.01.

FIG 8

Western blot analysis of pyrin S205 dephosphorylation in BMDMs. LPS-primed BMDMs were left uninfected or infected with the indicated Y. pseudotuberculosis 32777 ΔyopM strain for 90 or 180 min. After infection, lysates were processed with denaturing RIPA lysis buffer, collected, and subjected to Western blotting using antibodies to pSer205 of pyrin, total pyrin, or β-actin.