Extracts of Artemisia annua leaves and seeds mediate programmed cell death in Leishmania donovani

ABSTRACT Leishmania promastigote cells transmitted by the insect vector get phagocytosed by macrophages and convert into the amastigote form. During development and transformation, the parasites are exposed to various concentrations of reactive oxygen species, which can induce programmed cell death (PCD). We show that a mitochondrial peroxiredoxin (LdmPrx) protects Leishmania donovani from PCD. Whereas this peroxiredoxin is restricted to the kinetoplast area in promastigotes, it covers the entire mitochondrion in amastigotes, accompanied by dramatically increased expression. A similar change in the expression pattern was observed during the growth of Leishmania from the early to the late logarithmic phase. Recombinant LdmPrx shows typical peroxiredoxin-like enzyme activity. It is able to detoxify organic and inorganic peroxides and prevents DNA from hydroxyl radical-induced damage. Most notably, Leishmania parasites overexpressing this peroxiredoxin are protected from hydrogen peroxide-induced PCD. This protection is also seen in promastigotes grown to the late logarithmic phase, also characterized by high expression of this peroxiredoxin. Apparently, the physiological role of this peroxiredoxin is stabilization of the mitochondrial membrane potential and, as a consequence, inhibition of PCD through removal of peroxides.

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Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania

Eukaryotic Cell ◽

10.1128/ec.00123-07 ◽

2007 ◽

Vol 6 (10) ◽

pp. 1745-1757 ◽

Cited By ~ 91

Author(s):

Nancy Lee ◽

Sreenivas Gannavaram ◽

Angamuthu Selvapandiyan ◽

Alain Debrabant

Keyword(s):

Hydrogen Peroxide ◽

Cell Death ◽

Programmed Cell Death ◽

Leishmania Donovani ◽

Protozoan Parasite ◽

Protein Band ◽

Mrna Levels ◽

Enzymatic Assays ◽

Rich Domain ◽

Axenic Amastigotes

ABSTRACT In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single ∼42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and N α-tosyl-l-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.

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Racemoside A, an anti-leishmanial, water-soluble, natural steroidal saponin, induces programmed cell death in Leishmania donovani

Journal of Medical Microbiology ◽

10.1099/jmm.0.47114-0 ◽

2007 ◽

Vol 56 (9) ◽

pp. 1196-1204 ◽

Cited By ~ 50

Author(s):

Avijit Dutta ◽

Angana Ghoshal ◽

Debayan Mandal ◽

Nirup B. Mondal ◽

Sukdeb Banerjee ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Leishmania Donovani ◽

Chromatin Condensation ◽

Membrane Integrity ◽

Water Soluble ◽

Asparagus Racemosus ◽

Steroidal Saponin ◽

Significant Activity ◽

Major Health Problem

Leishmaniasis remains a major health problem of the tropical and subtropical world. The visceral form causes the most fatalities if left untreated. Dramatic increases in the rates of infection and drug resistance and the non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents. This study reports that racemoside A, a water-soluble steroidal saponin purified from the fruits of Asparagus racemosus, is a potent anti-leishmanial molecule effective against antimonial-sensitive (strain AG83) and -unresponsive (strain GE1F8R) Leishmania donovani promastigotes, with IC50 values of 1.15 and 1.31 μg ml−1, respectively. Incubation of promastigotes with racemoside A caused morphological alterations including cell shrinkage, an aflagellated ovoid shape and chromatin condensation. This compound exerts its leishmanicidal effect through the induction of programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide, loss of mitochondrial membrane potential culminating in cell-cycle arrest at the sub-G0/G1 phase, and DNA nicking shown by deoxynucleotidyltransferase-mediated dUTP end labelling (TUNEL). Racemoside A also showed significant activity against intracellular amastigotes of AG83 and GE1F8R at a 7–8-fold lower dose, with IC50 values of 0.17 and 0.16 μg ml−1, respectively, and was non-toxic to murine peritoneal macrophages up to a concentration of 10 μg ml−1. Hence, racemoside A is a potent anti-leishmanial agent that merits further pharmacological investigation.

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