<i>Aeromonas hydrophila</i> inhibits autophagy triggering cytosolic translocation of mtDNA which activates the pro-apoptotic caspase-1/IL-1β-nitric oxide axis in headkidney macrophages

Abstract

The molecular mechanisms underlying <i>Aeromonas hydrophila</i>-pathogenesis are not well understood. Using head kidney macrophages (HKM) of <i>Clarias gariepinus</i>, we previously reported the role of ER-stress in <i>A. hydrophila</i>-induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca²⁺ imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in <i>A. hydrophila-</i>infected HKM. CHOP promotes HKM apoptosis by inhibiting AKT activation and enhancing JNK signaling. Elevated mitochondrial ROS (mtROS) was recorded which declined significantly by ameliorating ER-stress and in the presence of ER-Ca²⁺ release modulators (2-APB and dantrolene) and mitochondrial-Ca²⁺ uptake inhibitor, Ru360, together suggesting the role of ER-mitochondrial Ca²⁺ dynamics in mtROS generation. Inhibiting mtROS production reduced HKM death implicating the pro-apoptotic role of mtROS in <i>A. hydrophila</i>-pathogenesis. The expression of autophagic proteins (LC3B, beclin-1, and atg 5) was suppressed in the infected HKM. Our results with autophagy-inducer rapamycin demonstrated that impaired autophagy favored the cytosolic accumulation of mitochondrial DNA (mtDNA) and the process depended on mtROS levels. Enhanced caspase-1 activity and IL-1β production was detected and transfection studies coupled with pharmacological inhibitors implicated mtROS/mtDNA axis to be crucial for activating the caspase-1/IL-1β cascade in infected HKM. RNAi studies further suggested the involvement of IL-1β in generating pro-apoptotic NO in <i>A. hydrophila</i>-infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating <i>A. hydrophila</i> pathogenesis. Based on our observations, we conclude that <i>A. hydrophila</i> induces ER-stress and inhibits mitophagy resulting in mitochondrial dysfunction which leads to mtROS production and translocation of mtDNA into cytosol triggering the activation of caspase-1/IL-1β-mediated NO production, culminating in HKM apoptosis.