The Yersinia virulence effector YopM binds caspase-1 to arrest inflammasome assembly and processing

Abstract

Inflammasome assembly activates caspase-1 and initiates the inflammatory cell death program pyroptosis, which is protective against numerous pathogens. Consequently, several pathogens, including the plague causing bacterium Yersinia pestis, avoid activating this pathway to enhance their virulence. However, bacterial molecules that directly modulate the inflammasome have yet to be identified. Examining the contribution of Yersinia type III secretion effectors to caspase-1 activation, we identified the leucine-rich repeat effector YopM as a potent antagonist of both caspase-1 activity and activation. YopM directly binds caspase-1, which both inhibits caspase-1 activity and sequesters it to block formation of the mature inflammasome. Caspase-1 activation antagonizes Yersinia survival in vivo, and consequently YopM inhibition of caspase-1 is required for Yersinia pathogenesis. Thus, a bacterium obstructs pyroptosis utilizing a direct mechanism of caspase-1 inhibition that is distinct from known viral or host inhibitors.

Figure 1. see also Figure S1. YopM prevents caspase-1 induction of pyroptosis

(A to E) Macrophages were infected with the Ypstb strains indicated (A to C) Cells with active caspase-1 were counted at the times indicated (A) or after 90 minutes of infection (B and C). (D) Caspase-1 immunoblot of infected cells indicates YopM prevents caspase-1 maturation. (E) ELISA shows release of IL-1β, confirmed by western blot for mature IL-1β, during YersiniaΔ infection but not during infection with YersiniaΔ expressing YopM in trans (+pYopM). (F) Lysosomal exocytosis, determined by immunofluorescence staining of surface LAMP1 on intact cells, was induced by infection and blocked by YopM. (G) Cellular lysis was measured from macrophages infected 2 hrs with Ypstb; lysis was caspase-1-dependent (indicating pyroptosis) and blocked by YopM. “ns”, p>0.05; “nd”, none detectable; data in A, B, C, E, F, and G are presented as mean +/ - SEM.

Figure 2. YopM is required in vivo for inhibition of caspase-1

(A) Four days post intra-peritoneal inoculation with 1000 CFU of Ypstb, few bacteria were recovered from the spleens of C57BL/ 6 infected with ΔyopM Ypstb, while this strain colonized casp1^-/- mice similar to fully virulent Ypstb; each point represents a single mouse. (B) WT Ypstb is lethal to both wild-type C57BL/ 6 mice (black) and casp1^-/- mice (gray). (C) However, only casp1^-/- mice are susceptible to ΔyopM Ypstb. Results are from combined experiments; asterisks indicate statistical differences by nonparametric Mann–Whitney U-test (titers, A) or Wilcoxon test (survival curves, B and C), *P < 0.05, **P < 0.005.

Figure 3. see also Figure S2. YopM is a pseudosubstrate inhibitor of caspase-1 activity

(A) Macrophages were challenged or treated with YersiniaΔ, Salmonella typhimurium, nigericin, or anthrax LT (10); retroviral transduction of YopM blocks caspase-1 activation in response to each of these stimuli; data are presented as mean +/ - SEM. (B) The conserved substrate recognition site bound by caspase-1 for selected known substrates and inhibitors, aligned to the similar sequence identified in YopM. The highly conserved aspartic acid is highlighted in red. (C) Model of YopM (blue, pdb: 1G9U) docking Caspase-1 (yellow, pdb: 1BMQ) via pseudosubstrate site (red). (D) Lysates from macrophages undergoing pyroptosis (infected with YersiniaΔ, lacking YopM) were incubated with 6xHis-YopM or 6xHis-YopM_271A as bait for pull-down and immunoblot detection of caspase-1. The sample loaded (L) contains both the 45kDa procaspase-1 as well as the cleaved p10 subunit indicative of mature caspase-1. Caspase-1 was detected in the wash volumes (W) with YopM_271A but not YopM; the bound fraction (B) contained caspase-1 only when YopM was used as bait. Enrichment of p10 over procaspase-1 in the YopM-bound (B) fraction compared with the loading (L) indicates YopM may have a higher affinity for mature caspase-1. (E) YopM, but not YopM_271A, blocks proteolysis of caspase-1 substrate YVAD-pNA.

Figure 4. see also Figure S3. YopM inhibits inflammasome formation and caspase-1 activation

Wild-type or casp1^-/- macrophages infected with Ypstb were fixed and protein localization visualized (A, C, E, G, I) and quantified (B, D, F, H) by immunofluorescence microscopy. Both NLRP3 (A and B) and the inflammasome adaptor protein ASC (C and D) rapidly form foci (arrowheads) in response to infection by Ypstb whether expressing YopM (+pYopM), inactive YopM (+pYopM_271A), or no YopM at all (YersiniaΔ) (blue, red, and green lines, respectively, in B, D, F, H). (E and F) In contrast, recruitment of caspase-1 to foci is blocked by YopM but not YopM_271A. (G and H) Caspase-1 is not activated in cells in which YopM can bind caspase-1. (I) In YopM-transduced, infected macrophages, YopM and caspase-1 colocalize. Representative confocal images are shown with macrophage nuclei visible by DNA staining (blue) and foci indicated with arrowheads; scale bars are 10 μm; data in B, D, F, and H are presented as mean +/ - SEM.