The <i>Yersinia pestis</i> Effector YopM Inhibits Pyrin Inflammasome Activation - Fig 4

<p>A) IL-1β was measured by ELISA in supernatants of LPS-primed BMDMs infected for 6 hours with <i>Y</i>. <i>pseudotuberculosis</i> 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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006035#ppat.1006035.ref033" target="_blank">33</a>]. 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.</p

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

<p>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 <i>Y</i>. <i>pestis</i> T3SS with inflammasome pathways.</p

The <i>Yersinia pestis</i> Effector YopM Inhibits Pyrin Inflammasome Activation - Fig 2

<p>LPS-primed BMDMs were infected with indicated <i>Y</i>. <i>pestis</i> 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 <i>Y</i>. <i>pestis</i> 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.</p

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

<p>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 <i>Y</i>. <i>pestis</i> 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 <i>Y</i>. <i>pestis</i> 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.</p

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

<p>IL-1β in supernatants from A) WT LPS-primed BMDMs infected with <i>Y</i>. <i>pestis</i> 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.</p