Elsevier

Free Radical Biology and Medicine

Volume 47, Issue 10, 15 November 2009, Pages 1494-1506
Free Radical Biology and Medicine

Original Contribution
Role of a differentially expressed cAMP phosphodiesterase in regulating the induction of resistance against oxidative damage in Leishmania donovani

https://doi.org/10.1016/j.freeradbiomed.2009.08.025Get rights and content

Abstract

Differentiation-coupled induction of resistance of Leishmania parasites to macrophage oxidative damage was shown to be associated with an increased cAMP response. This study explores the significance of the cAMP response in the parasite by identifying a differentially expressed cAMP phosphodiesterase (LdPDEA) and deciphering its role in regulating antioxidant machineries in the parasite. LdPDEA, a high KM class I cytosolic cAMP phosphodiesterase, was expressed maximally in log-phase promastigotes, but was significantly reduced in stationary-phase promastigotes and amastigotes. Chemical inhibition or silencing of PDEA conferred enhanced resistance to pro-oxidants in these cells and this led to studies on trypanothione biosynthesis and utilization, as trypanothione is one of the major modulators of antioxidant defense in kinetoplastidae. Despite enhanced arginase and ornithine decarboxylase activity, trypanothione biosynthesis seemed to be unaffected by PDEA blockage, whereas significant elevations in the expression of tryparedoxin peroxidase, ascorbate peroxidase, and tryparedoxin were detected, suggesting a definite shift of trypanothione-pool utilization bias toward antioxidant defense. Moreover, parasites that overexpressed PDEA showed reduced resistance to oxidative damage and reduced infectivity toward activated macrophages. This study reveals the significance of a cAMP phosphodiesterase in the infectivity of Leishmania parasites.

Section snippets

Parasite and cell culture

Pathogenic strains of L. donovani, AG83 (MHOM/IN/1983/AG83) and GE1 (MHOM/IN/89/GE1), were maintained in susceptible BALB/c mice and cultured as promastigotes in medium 199 (Invitrogen, Carlsbad, CA, USA) with Hanks’ salt containing Hepes (12 mM), l-glutamine (20 mM), 10% heat-inactivated FCS, 50 U/ml penicillin, and 50 μg/ml streptomycin. The promastigotes were obtained by culturing infected spleens in medium M199 for 5 days at 22°C. The adherent murine macrophage cell line RAW 264.7 was

Intracellular cAMP induces resistance to hydrogen peroxide and peroxynitrite

At the onset of mammalian infection, L. donovani promastigotes encounter a huge shift in temperature from 22°C in the insect gut to 37°C in the mammalian host and a shift of pH from 7.4 in sandfly gut to 5.5 in parasitophorous vacuoles of macrophages. We showed earlier that exposure to such temperature and pH renders the promastigotes more resistant to H2O2 and peroxynitrite (ONOO) and this was associated with an increase in intracellular cAMP level [13]. Similar to stress exposure, resistance

Discussion

Mining of the Leishmania genome revealed the presence of genes associated with the cAMP signaling pathway, and our previous work showed that differentiation conditions can induce resistance to oxidative damage via a cAMP-mediated response in L. donovani [13]. However, a defined participation of any of those genes in environmental sensing as well as in differentiation-coupled events is yet to be determined. The results of this study show that differentiation-coupled depletion of a cytosolic cAMP

Acknowledgments

This work was funded by the Department of Science and Technology and a Network Project grant (NWP 0038) from the Council of Scientific and Industrial Research (Government of India).

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    These authors contributed equally to this work.

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