Functional Involvement of Leishmania donovani Tryparedoxin Peroxidases during Infection and Drug Treatment

Abstract

The parasite causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.

Leishmania donovani parasite requires Atg8 protein for infectivity and survival under stress

“…A strain of L. donovani , BHU1260, was derived from the splenic aspirate of a VL patient at the Kala Azar Medical Centre of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India 44 . The culture was maintained in Medium 199 (M199) supplemented with 10% fetal calf serum (FCS) at 25 °C.…”

“…GFP-expressing cells could not be assessed for green fluorescence due to interference. Total intracellular ROS was measured by 2′,7′-dichlorodihydrofluorescein diacetate (H 2 -DCFDA) (Thermo Fisher Scientific; D399) as described earlier 44,49 (data not shown). Briefly, 5 × 10 6 cells were suspended in complete media and incubated with 2 μg·ml −1 CM-H 2 DCFDA for 15 min, and the desired treatments were carried out followed by fluorescence assay by using BMG black 96-well plates measured by using a microplate reader (CLARIOstar, BMG Labtech) at an excitation/emission at 530–10/590–10 nm.…”

“…Briefly, 5 × 10 6 cells were suspended in complete media and incubated with 2 μg·ml −1 CM-H 2 DCFDA for 15 min, and the desired treatments were carried out followed by fluorescence assay by using BMG black 96-well plates measured by using a microplate reader (CLARIOstar, BMG Labtech) at an excitation/emission at 530–10/590–10 nm. Mitochondria-generated superoxide was measured with MitoSOX™ Red (Thermo Fisher Scientific; M36008), a probe for detection of superoxides in the mitochondria as described previously 44 . About 5 × 10 6 parasites ml −1 were preincubated with 0.5 M MitoSOX™ Red in 1× PBS for 30 min at room temperature.…”

Leishmania donovaniInduces Autophagy in Human Blood–Derived Neutrophils

“…The hPMNs were pretreated with a cell-permeable ROS indicator H 2 DCFDA (25 mM for 45 min, Invitrogen) seeded onto BMG black 96well plate with cell density of 2 3 10 6 cells/well. Cells were exposed to different treatments after the baseline ROS reading was recorded with a CLARIOstar fluorometer (BMG Labtech, Ortenberg, Germany) with excitation/emission wavelength at 530-10/590-10 nm as described earlier (23). Postinfection, ROS kinetics was measured for 7 h duration at 15 min intervals.…”

The ultimate fate determinants of drug induced cell-death mechanisms in Trypanosomatids

“…Amphotericin B-resistant or potassium antimony-resistant isolates exhibit the up-regulation of cytoplasmic TXNPx in Leishmania species ( Andrade and Murta, 2014 ) thus indicating a portion of AmB treated parasites undergoing ferroptosis only or in conjunction with apoptosis-like death. When trypanosomatids are treated with the mitochondrial complex I inhibitor Rotenone, there is a dose-dependent increase in both cytoplasmic and mitochondrial TXNPx expression ( Das et al., 2017 ). Overall it appears that in trypanosomatids, targeted inhibition of peroxidases and tryparedoxins followed by mitochondrial dysfunction and unrepaired lipid peroxides result in ferroptosis mediated cell death.…”