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 Rhomodifying 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-1␤ (IL-1␤) 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 RhoAinactivating 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 ΔyopM Yersinia 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.

The pyrin inflammasome and the Yersinia effector interaction

“…YopE GAP and YopT protease activities were demonstrated to trigger the pyrin inflammasome by infecting LPS‐primed BMDMs with Yersinia strains in which the yopE or yopT genes were mutated in a yopM mutant background 16,17,18,47 . Introduction of mutations that inactivated the two genes or the catalytic activities of the gene products alone or in combination demonstrated that YopE GAP has a dominant role in triggering activation of pyrin (Figure 1, Step 12) 16,17,18,47 . Pyrin activation triggered by YopT protease activity (Figure 1, Step 13) can only be detected in the absence of YopE in LPS‐primed BMDMs infected with Y. pseudotuberculosis 16,18 .…”

“…Introduction of mutations that inactivated the two genes or the catalytic activities of the gene products alone or in combination demonstrated that YopE GAP has a dominant role in triggering activation of pyrin (Figure 1, Step 12) 16,17,18,47 . Pyrin activation triggered by YopT protease activity (Figure 1, Step 13) can only be detected in the absence of YopE in LPS‐primed BMDMs infected with Y. pseudotuberculosis 16,18 . No role for YopT in activating pyrin could be demonstrated in LPS‐primed BMDMs infected with Y. pestis 17,47 .…”

“…The concept that inactivation of RhoA triggers pyrin activation was established using toxins and effectors that introduce covalent modifications in or near the switch 1 region. Since non‐covalent inactivation of RhoA by YopE GAP and detachment of RhoA from plasma membrane by YopT protease also triggers pyrin activation, it is likely that any toxin or effector mechanism that prevents RhoA from activating PRK is sufficient to trigger linker dephosphorylation 6,16‐18 …”

“…The GDI‐like domain of YpkA can inhibit nucleotide exchange on RhoA and Rac1, leading to disruption of actin filaments 59 . Infection of LPS‐primed BMDMs with a Yersinia yopEyopTyopM mutant triggers activation of the pyrin inflammasome with delayed kinetics 18 . The possibility that YpkA GDI activity triggers delayed activation of pyrin remains to be investigated.…”

“…Novel insights into the regulation and function of pyrin can be gained through the understanding of its interactions with toxins and effectors of bacterial pathogens 6 . This may be best exemplified by the interaction of pyrin with Yersinia effectors 16‐19 . Yersinia spp.…”