Leishmania donovani Tryparedoxin Peroxidases: functional involvement during infection and drug treatment

21 The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal 22 disease. These parasites survive within mammalian macrophages and express a 23 unique set of enzymes, the tryparedoxin peroxidases for their defense against oxidative 24 stress generated by the host. In this study, we demonstrate a differential role of two 25 distinct enzymes, the mitochondrial (mTXNPx) and the cytosolic tryparedoxin 26 peroxidase (cTXNPx) in defending the parasites against mitochondrial and exogenous 27 oxidative stress during infection and drug treatment. Our findings indicate a 28 comparatively higher increase in cTXNPx expression in response to exogenous 29 oxidative stress and a relatively higher elevation of mTXNPx expression in response to 30 mitochondrial or endogenous stress created by respiratory chain complex inhibitors. 31 Overexpression of cTXNPx in the Leishmania show improved protection to exogenous 32 stress and enhanced protection to mitochondrial stress in parasites overexpressing 33 mTXNPx. Further, parasites overexpressing cTXNPx infect host cells with increased 34 efficiency at early hours of infection as compared to control parasites or parasites 35 overexpressing mTXNPx. The mTXNPx overexpressing parasites sustain higher 36 infection at later hours. Higher mTXNPx expression occurred in wild type parasites on 37 exposure to miltefosine while treatment with antimony elevated cTXNPx expression. 38 Parasites resistant to miltefosine or antimony demonstrated increased expression of 39 mTXNPx as well as cTXNPx. In summary, this study provides evidence of distinct role 40 of the two enzymes defined by virtue of their localization during infection and drug 41 treatment. 42 This study shows a new finding of the ability of the mTXNPx to regulate oxidative stress generated by mitochondrial toxins more efficiently than cTXNPx, whereas, cTXNPx was more competent in dealing with exogenous oxidative stress as compared to mTXNPx. Further, the findings show increase of early infection rates when cells were equipped with larger quantities of cTXNPx as compared to mTXNPx. Importantly, anti-leishmanial drugs like PAT and miltefosine showed different efficacy with increased quantities of the enzymes where presence of excess mTXNPx made the parasites less sensitive to miltefosine while high cTXNPx contributed resistance to PAT. to other parts of the cells. Knowledge on the relative importance of enzymes as a function of their location is important regarding the extent of utility of the enzymes in a protective role. This may further give clues to successful designing of drugs targeting the dual antioxidant systems of parasite. This study presents a new finding of a differential response to specific kind of stress that determines the functional outcome. It is understood that correct subcellular localization of proteins is critical as it defines the physiological context of the protein function. In that setting, mitochondrial localization of a peroxidase is very essential as mitochondria are the cradle of ROS generation. The extent of ROS production upon respiratory chain inhibition are different for complexes I, II, III and IV (20). Small inhibition of complex I is sufficient to increase ROS production, whereas about 70% inhibition could be tolerated by complex III and IV without an increase in ROS formation. Our previous studies have shown that inhibition of complexes I, II and III generate sufficient ROS to induce loss of mitochondrial function in the Leishmania culminating in cell death (20). The relatively higher expression of mTXNPx in WT cells as compared to cTXNPx when respiratory chain was inhibited, suggested a location specific defensive response of the cell to combat peroxides and their products. This was supported by observations of improved ability to withstand mitochondrial stress by parasites overexpressing mTXNPx as compared to WT and cTXNPx overexpressing cells. This was clear evidence of a mitochondria centric stress response when mitochondrial toxins disrupted the functioning of the organelle. The data of cell survival indicated a better endurance post mitochondrial in parasites overexpressing mTXNPx as compared to cTXNPx substantiating the that localization favorably protected the parasite. Since mitochondria