Aeromonas hydrophila 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 Aeromonas hydrophila-pathogenesis are not well understood. Using head kidney macrophages (HKM) of Clarias gariepinus, we previously reported the role of ER-stress in A. hydrophila-induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca²⁺ imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in A. hydrophila-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 A. hydrophila-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 A. hydrophila-infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating A. hydrophila pathogenesis. Based on our observations, we conclude that A. hydrophila 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.

KEYWORDS:

• Aeromonas hydrophila
• head kidney macrophage
• er-stress
• mtROS
• mtDNA
• autophagy
• apoptosis
• caspase-1
• IL-1β

Acknowledgments

The authors are grateful to S. Barik, Cleveland State University for helpful discussions and critically analyzing the manuscript. We thank Chaitali Banerjee for help in immunoblot studies and Tushar K. Maiti and Manjula Kalia, Regional Centre for Biotechnology (RCB), Faridabad and Dhiraj Kumar, ICGEB, New Delhi for help in protein transfection and apoptosis studies.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceived and designed the experiments: MK, AS, PD, SM, AR. Performed the experiments: MK, AS, PD, AR. Analyzed the data: MK, AS, PD, SM. Contributed reagents/materials/analysis tools: SM. Wrote the paper: MK, AS, SM.

Data availability:

Data sharing not applicable – no new data generated.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

The research work was partially supported by Recurring Research Grant (RRG), South Asian University [RRG-2019]. MK and PD were supported by UGC-NET Fellowship (Government of India) and AS was supported by DBT Fellowship (Govt. of India). AR was supported by Dr DS Kothari Post Doctoral Fellowship, UGC (Government of India). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.