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

Manmohan Kumar; Asha Shelly; Priyanka Dahiya; Atish Ray; Shibnath Mazumder

doi:10.1080/21505594.2021.2018767

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

Virulence. 2022 Dec;13(1):60-76. doi: 10.1080/21505594.2021.2018767.

Authors

Manmohan Kumar¹, Asha Shelly¹, Priyanka Dahiya¹, Atish Ray¹, Shibnath Mazumder^{1

2}

Affiliations

¹ Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
² Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.

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; IL-1β; apoptosis; autophagy; caspase-1; er-stress; head kidney macrophage; mtDNA; mtROS.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aeromonas hydrophila* / genetics
Animals
Apoptosis
Autophagy
Caspase 1 / metabolism
Cytosol / metabolism
DNA, Mitochondrial / metabolism
Interleukin-1beta / metabolism*
Macrophages
Mitochondria / metabolism
Nitric Oxide* / metabolism
Reactive Oxygen Species / metabolism

Substances

DNA, Mitochondrial
Interleukin-1beta
Reactive Oxygen Species
Nitric Oxide
Caspase 1

Grants and 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.