Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP–PKA signaling axis for its intra‐macrophage survival during infection

Abstract

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by~1.54 ± 0.35, and~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by~5.7 ± 0.35,~2.57 ± 0.56,~4.7 ± 0.36, and 2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and 25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less Biotechnology, Ministry of Science and Technology, Grant/Award Number: DBT/ JRF/16/AL/986 infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

Identification of Philaster apodigitiformis in aquaculture and functional characterization of its β-PKA gene and expression analysis of infected Poecilia reticulata

“…The PKA gene has been reported in previous studies to have the ability to participate as a second messenger and to help Plasmodium invade the host cell (Flueck et al ., 2019 ; Patel et al ., 2019 ; Kumar et al ., 2021 ). In the previous study, we found a significant upregulation of the expression level of β-PKA gene by the transcriptome analysis after the pre-infestation of P. apodigitiformis on ornamental fish.…”

“…Previous studies show that protein kinase A (PKA) is a prominent member of the AGC kinase family. β-PKA controls a variety of cellular metabolism processes, including cell growth, gene expression and metabolism (Leroux et al ., 2018 ; Kumar et al ., 2021 ). Therefore, β-PKA can play an important role in various infection and toxicological processes.…”