Leishmania donovani cathepsin B and antioxidant proteins: role in Leishmania survival and interaction with host macrophages.

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Leishmania parasites are causative agents of leishmaniasis in humans. Leishmania donovani complex, the etiological agents of visceral leishmaniasis, account for over 500,000 new cases and 59,000 deaths every year. Leishmania has evolved numerous mechanisms to evade the host immune response and survive within macrophages. It modulates the immune response by inhibiting antigen presentation, suppression of cytokine production and induction of immunosuppressive molecules. Transforming growth factor beta (TGF-ß) is one of the major pro-inflammatory cytokines produced during Leishmania - host macrophage interact to escape microbicidal activities of the host macrophage. Leishmania cathepsin B cysteine protease is implicated in this process. We have shown that Leishmania Cathepsin B cleaves and activates pre-TGF-ß1 in vitro. However, the effect of cathepsin B on TGF-ß in vivo has not been studied. We have also demonstrated that disruption of cathepsin B gene results in the modulation of L. donovani proteome primarily affecting secreted proteins involved in oxidation-reduction suggesting cathepsin B role in exosome based secretion of proteins and antioxidant defense system. Leishmania possesses iron superoxide dismutases (FeSODs) and peroxidoxins to detoxify reactive oxygen species (ROS) for its survival within macrophages. The mechanism by which cathepsin B affects exosome based secretion of Leishmania virulence factors (including proteins involved in antioxidant defense system) is not clear. Furthermore, the role of the Leishmania antioxidant proteins, FeSODs and peroxidoxins, in the survival during host- parasite interactionand the mechanism of the antioxidant defense system is not well understood. We have shown that superoxide dismutase-A (FeSODA) and peroxidoxin-4 (Pxn4) are targeted to the mitochondria and protects Leishmania parasites invitro from mitochondrial-derived ROS damage and programmed cell-death. However, the mechanism by which mitochondria is protected from ROS damage which will result in programmed cell death is unknown. Further studies on the mechanism(s) and role of FeSODA and Pxn4 in survival and programmed cell-death invivo would provide more insights on the mechanism(s) of Leishmania-host interaction . Thus, understanding the basic mechanism(s) by which cathepsin B affects TGF-ß and exosome based secretion of Leishmania proteins in modulating macrophage signaling as well as role of FeSOD and Pxn4 in Leishmania programmed cell death will shed light on host-parasite interaction and will have implication for microbes to modulate macrophage signaling using similar strategies. We hypothesize that: (1) Leishmania cathepsin B plays role in survival in the host by targeting TGF-ß and in secretion of Leishmania virulence factors including antioxidant proteins to modulate macrophage signaling and function. (2) Both FeSODA and Pxn4 play important functional role in parasite survival in the host by protecting mitochondria from ROS damage and thus programmed cell-death of Leishmania. The specific objectives of this proposal are to: (1) Study the role of L. donovani cathepsin B in survival in the host by targeting TGF-ß. (2) Determine the effect of L. donovani cathepsin B on Leishmania secreted proteins and their role in host-parasite interaction. (3) Generate FeSODA and Pxn4 null mutant as well as complemented parasites and assess their role in parasite survival and host-parasite interactions.