Figure 1.
Infection with V. parahaemolyticus induces caspase-1 activation, IL-1β maturation and pyroptosis via NLRP3 and NLRC4 inflammasomes.
BMMs from wild-type, ASC-deficient (Asc−/−), caspase-1-deficient (Casp1−/−), NLRP3-deficient (Nlrp3−/−), NLRC4-deficient (Nlrc4−/−), or NLRP3/NLRC4-double deficient (Nlrp3−/−Nlrc4−/−) mice were primed with LPS (1 µg/ml; 3 hr) and infected with wild-type V. parahaemolyticus (Vp). A. Phase contrast images of wild-type BMMs either uninfected (-) or 3 hour post-infection (hpi). Representative damaged macrophages are shown. Bar, 20 µm. B and E. The activation of caspase-1 and IL-1β processing in infected BMMs were analyzed using immunoblotting with anti-caspase-1 or anti-IL-1β antibody. C and F. IL-1β secretion from the infected BMMs into the culture supernatants at 3 hpi. was analyzed using an ELISA. *p<0.05. D and G. Culture supernatants from infected BMMs were analyzed for LDH release. Data are presented as the means ± SD of triplicate samples. *p<0.05.
Figure 2.
Thermostable direct hemolysins (TdhA and TdhS) and T3SS1 are essential for the induction of caspase-1 activation and cell death.
BMMs from wild-type mice were primed with LPS and infected with WT V. parahaemolyticus or its respective isogenic mutants. Cell lysates and culture supernatants were harvested at indicated times post infection. A. Activation of caspase-1 and IL-1β processing in infected wild-type BMMs were analyzed using immunoblotting with anti-caspase-1 or anti-IL-1β antibody. B. IL-1β secretion into culture supernatants from infected BMMs at 3 hpi. was analyzed using an ELISA. Data are presented as the means ± SD of triplicate samples. *p<0.05. C. Culture supernatants from infected BMMs were analyzed for LDH release. Data are presented as the means ± SD of triplicate samples.
Figure 3.
TDHs trigger caspase-1 activation via NLRP3 inflammasome, whereas T3SS1 triggers both NLRP3 and NLRC4 inflammasomes.
BMMs from wild-type, ASC-deficient (Asc−/−), caspase-1-deficient (Casp1−/−) NLRP3-deficient (Nlrp3−/−), NLRC4-deficient (Nlrc4−/−), or NLRP3/NLRC4-double deficient (Nlrp3−/−Nlrc4−/−) mice were primed with LPS and infected with V. parahaemolyticus mutant, ΔvcrD1 (TDHs intact) or ΔtdhAS (T3SS1 intact). A, D, G and J. Activation of caspase-1 and IL-1β processing in infected BMMs were analyzed using immunoblotting with anti-caspase-1 or anti-IL-1β antibody. B, E, H and K. IL-1β secretion into culture supernatants from infected BMMs at 3 hpi. was analyzed using an ELISA. Data are presented as the means ± SD of triplicate samples. *p<0.05. C, F, I and L. Culture supernatants from infected BMMs were analyzed for LDH release. Data are presented as the means ± SD of triplicate samples. *p<0.05.
Figure 4.
T3SS1 effectors suppress NLRC4-mediated inflammasome activation.
BMMs from wild-type, NLRP3-deficient (Nlrp3−/−), or NLRC4-deficient (Nlrc4−/−) mice were primed with LPS and infected with V. parahaemolyticus mutant, ΔtdhAS or ΔtdhASΔh1in which the effectors' genes are deleted. A. Schematic representation of gene structure in the h1 region of T3SS1. B. Activation of caspase-1 and IL-1β processing in infected BMMs were analyzed using immunoblotting with anti-caspase-1 or anti-IL-1β antibody. C. IL-1β secretion into culture supernatants from infected BMMs at 3 hpi. was analyzed using an ELISA. Data are presented as the means ± SD of triplicate samples. *p<0.05. D. Culture supernatants from infected BMMs were analyzed for LDH release. Data are presented as the means ± SD of triplicate samples. *p<0.05.
Figure 5.
The effectors VopQ and VopS are involved in the suppression of inflammasome activation.
A, B and C. BMMs from NLRP3-deficient (Nlrp3−/−) mice were primed with LPS and infected with ΔtdhAS or its respective isogenic mutants as indicated. A. Activation of caspase-1 and IL-1β processing in infected BMMs were analyzed using immunoblotting with anti-caspase-1 or anti-IL-1β antibody. B and C. IL-1β secretion into culture supernatants from infected BMMs at 3 hpi. was analyzed using an ELISA. Data are presented as the means ± SD of triplicate samples. *p<0.05.
Figure 6.
VopQ-mediated autophagy interferes with NLRC4 inflammasome activation.
A. BMMs from wild-type or NLRP3-deficient (Nlrp3−/−) mice were infected with ΔtdhAS or ΔtdhASΔvopQ mutants for one hour, and then immunostained with FITC-labeled anti-LC3 antibody (green) and Cy5-labeled anti-V. parahaemolyticus antibody (red). The merged image and differential interference contrast (DIC) were shown. Bar, 10 µm. B. BMMs were infected with bacteria (1 h) or treated with rapamycin as a control (2 h) in the absence or presence of bafilomycin A1. The cells were analyzed by immunoblotting with anti-LC3 antibody to evaluate the conversion of endogenous LC3-I to II or with anti-β-actin antibody. The relative intensity of LC3-II was calculated by normalizing the untreated control to a relative intensity of 1 and then dividing subsequent LC3-II band intensity by actin band intensity using densitometry. C. Knockdown efficiency of Atg5 by shRNA in NLRP3-deficient BMMs was analyzed by immunoblotting with anti-Atg5 antibody. D. Effect of knockdown of Atg5 on rapamycin-mediated LC3-I to II conversion in NLRP3-deficient BMMs. E. Effect of knockdown of Atg5 on caspase-1 activation and IL-1β processing in infected NLRP3-deficient BMMs infected with indicated bacteria. F. Effect of knockdown of Atg5 on IL-1β release from infected NLRP3-deficient BMMs at 3 hpi. was analyzed using an ELISA. Data are presented as the means ± SD of triplicate samples. *p<0.05.
Figure 7.
Inactivation of Cdc42 by VopS is involved in suppression of NLRC4 inflammasome activation.
A and B. Activated endogenous Cdc42 was analyzed by GST-PAK PBD pull-down and immunoblotting with anti-Cdc42 antibody. A. The cell lysates of NLRP3-deficient BMMs infected with ΔtdhAS or ΔtdhASΔvopS mutants for one hour were loaded with GDP or GTP-γS. B. Cell lysates of NLRP3-deficient BMMs infected with ΔtdhAS, ΔtdhASΔvopS mutants complemented by WT vopS or vopS H348A for 1 h were loaded with or without GTP-γS. C. Effect of VopS H348A mutant on caspase-1 activation and IL-1β processing in infected NLRP3-deficient BMMs with ΔtdhASΔvopS mutant or complemented strains by wild-type vopS or vopS H348A. D. Effect of VopS H348A mutant on release of IL-1β from infected NLRP3-deficient BMMs with ΔtdhASΔvopS mutant or complemented strains as in C. Data are presented as the means ± SD of triplicate samples. *p<0.05. E. Effect of VopS H348A mutant on release of IL-1β from infected NLRP3-deficient BMMs with ΔtdhASΔvopQS mutant or complemented strains as in C. Data are presented as the means ± SD of triplicate samples. *p<0.05.
Figure 8.
VopQ and VopS inhibit speck formation by ASC.
A. BMMs from NLRP3-deficient (Nlrp3−/−) mice were infected with indicated bacteria for one hour, and then immunostained with FITC-labeled anti-ASC antibody (green) and Cy5-labeled anti-V. parahaemolyticus antibody (red). The merged images were shown. Bar, 10 µm. B. Quantitative data of number of speck-forming macrophages in A. Data are presented as the means ± SD of triplicate samples. *p<0.05.