Figures & data
Figure 1. A. hydrophila-induced PI3K/PLC axis activates ER-stress. HKM pre-incubated with 4-PBA, LY-294002, U73122, YCG063, and Antimycin A were infected with or without A. hydrophila and chop expression studied at 2 h p.i. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+4-PBA+B, HKM pre-incubated with 4-PBA infected with A. hydrophila; HKM+LY-294002 + B, HKM pre-incubated with LY-294002 infected with A. hydrophila; HKM+U73122 + B, HKM pre-incubated with U73122 infected with A. hydrophila; HKM+YCG063 + B, HKM pre-incubated with YCG063 infected with A. hydrophila, HKM+Antimycin A, HKM pre-incubated with Antimycin A.
Figure 2. A. hydrophila-induced ER-stress induces HKM apoptosis via JNK-Akt signaling pathway. HKM pre-incubated with SP600125, 124,005 or transfected with sc-siRNA or chop-siRNA were infected with A. hydrophila and at 24 h p.i. (a) phosphorylation of JNK, (b) levels of total and phospho-Akt, and (c) HKM apoptosis was studied. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+SP600125 + B, HKM pre-incubated with SP600125 infected with A. hydrophila; HKM+124005 + B, HKM pre-incubated with 124,005 infected with A. hydrophila; HKM+sc-siRNA, HKM transfected with sc-siRNA (scrambled siRNA); HKM+chop-siRNA+B, HKM transfected with chop-siRNA infected with A. hydrophila.
Figure 3. A. hydrophila induces pro-apoptotic mtROS production. HKM pre-incubated with Antimycin A, YCG063, 4-PBA, 2-APB, Dant, Ru360, Cyt D, heat-killed bacteria and Rapa were infected with or without A. hydrophila and (a) changes in mtROS levels were studied at 4 h p.i. and (b) HKM apoptosis was studied at 24 h p.i. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+YCG063 + B, HKM pre-incubated with YCG063 infected with A. hydrophila; HKM+4-PBA+B, HKM pre-incubated with 4-PBA infected with A. hydrophila; HKM+2-APB +B, HKM pre-incubated with 2-APB infected with A. hydrophila; HKM+Dant+B, HKM pre-incubated with dantrolene infected with A. hydrophila; HKM+Ru360 + B, HKM pre-incubated with Ru360 infected with A. hydrophila; HKM+Cyt D + B, HKM pre-incubated with Cyt D infected with A. hydrophila; HKM+heat-killed B, HKM infected with heat-killed A. hydrophila; HKM+Rapa+B, HKM pre-incubated with rapamycin infected with A. hydrophila, HKM+Antimycin A, HKM pre-incubated with Antimycin A.
Figure 4. A. hydrophila triggers translocation of mtDNA via suppression of autophagy. (a) HKM were pre-incubated with Antimycin A, YCG063, CsA, Rapa, or transfected with DNase I and heat-inactivated DNase I were infected with or without A. hydrophila and presence of mtDNA in cytosol was studied at 1 h p.i. (b) HKMs were infected with A. hydrophila and expression of autophagic proteins (beclin-1, atg 5, and LC3B) were studied at by immunoblotting. (c) HKM incubated with rapamycin infected with A. hydrophila and percentage HKM viability was studied at 24 h p.i. using trypan blue dye exclusion method. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). ns indicates not significant between indicated groups. HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+YCG063 + B, HKM pre-incubated with YCG063 infected with A. hydrophila; HKM+CsA+B, HKM pre-incubated with Cyclosporin A infected with A. hydrophila; HKM+Rapa+B, HKM pre-incubated with rapamycin infected with A. hydrophila; HKM+DNase I + B, HKM transfected with DNase I infected with A. hydrophila; HKM+heat-inactivated DNase I + B, HKM transfected with heat-inactivated DNase I infected with A. hydrophila; HKM+Antimycin A, HKM pre-incubated with Antimycin A.
Table 2. List of RT-qPCR primer sequences
Figure 5. Cytosolic mtDNA triggers activation of pro-apoptotic caspase-1 in A. hydrophila-infected HKM. (a) HKMs pre-incubated with Z-YVAD-FMK, 4-PBA, YCG063 or transfected with mtDNA, DNase I, heat-inactivated DNase I were infected with or without A. hydrophila and caspase-1 activity studied at 12 h p.i. (b) HKMs incubated with Z-YVAD-FMK were infected with A. hydrophila and HKM apoptosis studied at 24 h p.i. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+Z-YVAD-FMK+B, HKM pre-incubated with Z-YVAD-FMK infected with A. hydrophila; HKM+4-PBA+B, HKM pre-incubated with 4-PBA infected with A. hydrophila; HKM+YCG063 + B, HKM pre-incubated with YCG063 infected with A. hydrophila; HKM+mtDNA, HKM transfected with mtDNA; HKM+DNase I + B, HKM transfected with DNase I infected with A. hydrophila; HKM+heat-inactivated DNase I + B, HKM transfected with heat-inactivated DNase I infected with A. hydrophila.
Figure 6. A. hydrophila-induced mtDNA/caspase-1 axis triggers IL-1β production. HKM pre-incubated with Z-YVAD-FMK, 4-PBA, YCG063 or transfected with sc-siRNA, il1b-siRNA, DNase I, heat-inactivated DNase I, mtDNA were infected with or without A. hydrophila and production of IL-1β protein was measured at 24 h p.i. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). ns indicates not significant between indicated groups. HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+Z-YVAD-FMK+B, HKM pre-incubated with Z-YVAD-FMK infected with A. hydrophila; HKM+4-PBA+B, HKM pre-incubated with 4-PBA infected with A. hydrophila; HKM+YCG063 + B, HKM pre-incubated with YCG063 infected with A. hydrophila; HKM+sc-siRNA, HKM transfected with sc-siRNA; HKM+sc-siRNA+B, HKM transfected with sc-siRNA infected with A. hydrophila, HKM+il1b-siRNA+B, HKM transfected with il1b-siRNA infected with A. hydrophila; HKM+DNase I + B, HKM transfected with DNase I infected with A. hydrophila; HKM+heat-inactivated DNase I + B, HKM transfected with heat-inactivated DNase I infected with A. hydrophila; HKM+mtDNA, HKM transfected with mtDNA.
Figure 7. IL-1β induces activation of pro-apoptotic inos/NO axis in A. hydrophila-infected HKM. HKM transfected with sc-siRNA, il1b-siRNA, inos-siRNA or pre-incubated with L-NIL, Z-DEVD-FMK and infected with A. hydrophila and at 24 h p.i., (a) HKM apoptosis, (b) the expression of inos mRNA was studied, (c) nitrite levels were measured, and (d) caspase-3 activity was studied at 24 h p.i. Vertical bars represent mean ± SE (n = 3). Asterisk indicates significant difference between indicated groups (*p < 0.05). HKM, uninfected HKM; HKM+B, HKM infected with A. hydrophila; HKM+L-NIL+B; HKM pre-incubated with L-NIL and infected with A. hydrophila; HKM+Z-DEVD-FMK+B, HKM pre-incubated with Z-DEVD-FMK and infected with A. hydrophila; HKM+sc-siRNA, HKM transfected with sc-siRNA; HKM+sc-siRNA+B, HKM transfected with sc-siRNA infected with A. hydrophila; HKM+il1b-siRNA+B, HKM transfected with il1b-siRNA infected with A. hydrophila; HKM+inos-siRNA+B, HKM transfected with inos-siRNA infected with A. hydrophila.
Figure 8. Overview of the study. A. hydrophila-induced activation of PI3K/PLC axis triggers ER-stress leading to downstream phosphorylation of JNK and consequent inhibition of Akt phosphorylation. ER-stress instigates mtROS production leading to cytosolic translocation of mtDNA via suppression of autophagy. The cytosolic mtDNA activates inflammatory caspase-1/IL-1β axis leading to NO-mediated apoptosis of A. hydrophila-infected HKM.
Table 1. List of siRNA sequences
