The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation

Abstract

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.

Fig 1. The Y. pestis effector YopM suppresses a different IL-1β-producing pathway than the one triggered by the needle/translocon through NLRP3 and NLRC4.

IL-1β in supernatants from A) WT LPS-primed BMDMs infected with Y. pestis Yop mutant strains, B) BMDMs of indicated genotypes infected with ΔT3SSe, or C) LPS-primed BMDMs infected with KIM5 and ΔYopM were measured by ELISA 6 hrs p.i. (MOI 10). D) Cell death was assayed by LDH release in LPS-primed BMDMs infected with indicated strains at 6 hrs p.i. (MOI 10). Figures are representative of three or more experiments. E) Total protein from LPS-primed BMDMs infected with indicated strains (combined cell lysate and supernatant) was separated by SDS-PAGE and analyzed by Western Blot for IL-1β and caspase-1. F) Mice of indicated genotypes were injected s.c. with 160 CFU of KIM1001ΔM/J and monitored for survival past 21 days. P value for survival comparisons reflect differences between WT (n = 11) or NLRP3 KO (n = 11) and IL-18 (n = 6), Asc KO (n = 14). Shown is mean plus s.d. for triplicate wells. ND: not detected. A-E are representative of three experiments or more, F representative of two experiments performed. * p<0.05, **p<0.01, ***p<0.001.

Fig 2

LPS-primed BMDMs were infected with indicated Y. pestis strains for 6 hours; A) IL-1β, B) IL-18, and C) TNFα were measured in supernatants by ELISA 6 hrs p.i. (MOI 10); D) Total protein from LPS-primed BMDMs infected with indicated strains (combined cell lysate and supernatant) was separated by SDS-PAGE and analyzed by Western Blot for IL-1β and caspase-1. E) Cell death was assayed by LDH release in LPS-primed BMDMs infected with indicated strains at 6 hrs p.i. (MOI 10). F) Expression of Pyrin and Pro-IL-1β mRNA was measured by RT-PCR at 1, 3, 5, or 7 hours after addition of 100ng/mL LPS to WT BMDMs. G) WT C57Bl/6 (n = 12), Pyrin KO (n = 10), NLRP3 KO (n = 7) or IL-18 KO (n = 8) or H) WT (n = 9), Pyrin KO (n = 10) or caspase-1/11 KO (n = 8) mice were infected s.c. with Y. pestis KIM1001 ΔYopM/J (150 CFU) and monitored for survival up to 21 days. G, H): P value reflects comparison of WT vs Pyrin KO, NLRP3 vs Pyrin KO, WT vs IL-18 KO or WT vs caspase-1/11 KO. Figures are representative of three or more experiments, G, F are representative of two experiments. Shown is mean plus s.d. for triplicate wells. * p<0.05, **p<0.01, ***p<0.001.

Fig 3

A-F,E,G) IL-1β was measured by ELISA in supernatants of LPS-primed BMDMs infected for 6 hours with indicated bacterial strains, or D) cell death was measured by LDH release at 6 hours p.i. (MOI 10). Figures are representative of three or more experiments. Shown is mean plus s.d. for triplicate wells. * p<0.05, **p<0.01, ***p<0.001.

Fig 4

A) IL-1β was measured by ELISA in supernatants of LPS-primed BMDMs infected for 6 hours with Y. pseudotuberculosis IP2666, including strains expressing YopM with partial deletions (MOI 10). The numbers in the YopM protein refer to different leucine-rich repeat (LRR) domains of YopM, and C-term indicates the C-terminal end. RecM indicates reconstitution (rec) of IP2666 ΔYopM with variants of YopM, as shown in the figure. C7 and C8 are two different triple alanine substitutions near the C-terminus of YopM [33]. B) Total protein from LPS-primed BMDMs infected with indicated strains (combined cell lysate and supernatant) was separated by SDS-PAGE and analyzed by Western Blot for IL-1β and caspase-1. Figures are representative of two independent experiments. Shown is mean plus s.d. for triplicate wells. * p<0.05, **p<0.01, ***p<0.001.

Fig 5. YopM maintains an inhibitory phenotype in human PBMCs, and in a human THP-1 cell line overexpressing YFP-Pyrin.

Co-IP pulldown in these cells as well as mouse BMDMs indicate YopM interaction with Pyrin, Rsk1, Pkn1, and Iqgap1. A) PBMCs were isolated from healthy human donor blood and infected at MOI 10 with indicated Y. pestis strains without priming. At 6 hours p.i. supernatant was collected for IL-1β detection by ELISA. B) Cultured YFP-Pyrin THP-1 cells were differentiated with 100nM Vitamin D3 for 48–72 hours, and infected with indicated Y. pestis strains at MOI 10. Shown is IL-1β assayed from supernatants by ELISA at 6 hrs p.i. Figures are representative of three or more experiments. Shown is mean plus s.d. for triplicate wells. * p<0.05, **p<0.01, ***p<0.001. C-D) Shown are Western blot results of co-IP with anti-YopM using C) Vitamin D3-differentiated, unprimed YFP-Pyrin cells or D) LPS-primed BMDMs after infection with the indicated strains at MOI 10 for 3 hours. Bead-bound protein and lysates were separated by SDS-PAGE and analyzed by Western Blot for the proteins indicated.

Fig 6. YopM prevents the formation of Pyrin-dependent but not NLRP3-dependent Asc complexes.

A) HEK293T cells stably expressing Asc-YFP were transfected with pCDNA3-Pyrin, pRBH-YopM, or both constructs together. B) pCDNA3-NLRP3 and respective empty vectors were used as positive and negative controls. Asc speckles were visualized, quantified, and normalized to cell number. Figures are representative of three or more experiments. Shown is mean plus s.d. for triplicate fields quantified. * p<0.05, **p<0.01, ***p<0.001. C) Proposed model integrating the major interactions of the Y. pestis T3SS with inflammasome pathways.