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

利什曼原虫是人类利什曼病的病原体。杜氏利什曼原虫复合体是内脏利什曼病的病原体，每年有50多万新发病例和59，000例死亡。利什曼原虫已经进化出许多机制来逃避宿主免疫反应并在巨噬细胞内存活。它通过抑制抗原呈递、抑制细胞因子产生和诱导免疫抑制分子来调节免疫应答。转化生长因子β（TGF-β）是利什曼原虫-宿主巨噬细胞相互作用以逃避宿主巨噬细胞的杀微生物活性期间产生的主要促炎细胞因子之一。利什曼原虫组织蛋白酶B半胱氨酸蛋白酶参与该过程。我们已经证明利什曼原虫组织蛋白酶B在体外切割并激活前TGF-β 1。然而，尚未研究组织蛋白酶B对体内TGF-β的影响。我们还证明了组织蛋白酶B基因的破坏导致L. donovani蛋白质组主要影响参与氧化-还原的分泌蛋白，提示组织蛋白酶B在基于外泌体的蛋白质分泌和抗氧化防御系统中的作用。利什曼原虫具有铁超氧化物歧化酶（FeSOD）和过氧化物酶，以解毒活性氧（ROS），使其在巨噬细胞内存活。组织蛋白酶B影响利什曼原虫毒力因子（包括参与抗氧化防御系统的蛋白质）基于外泌体的分泌的机制尚不清楚。此外，利什曼原虫抗氧化蛋白FeSOD和过氧化物酶在宿主-寄生虫相互作用期间的存活中的作用以及抗氧化防御系统的机制尚不清楚。我们已经证明，超氧化物歧化酶-A（FeSODA）和过氧化物酶-4（Pxn 4）靶向线粒体，并保护利什曼原虫体外免受来自线粒体的ROS损伤和程序性细胞死亡。然而，保护线粒体免受ROS损伤的机制尚不清楚，ROS损伤将导致程序性细胞死亡。进一步研究FeSODA和Pxn 4在体内存活和程序性细胞死亡中的作用和机制将为利什曼原虫与宿主相互作用的机制提供更多的见解。因此，理解组织蛋白酶B影响TGF-β和基于外泌体的利什曼原虫蛋白分泌调节巨噬细胞信号传导的基本机制以及FeSOD和Pxn 4在利什曼原虫程序性细胞死亡中的作用将阐明宿主-寄生虫相互作用，并将暗示微生物使用类似策略调节巨噬细胞信号传导。（1）利什曼原虫组织蛋白酶B通过靶向TGF-β和分泌利什曼原虫毒力因子（包括抗氧化蛋白）以调节巨噬细胞信号传导和功能而在宿主中的存活中起作用。(2)FeSODA和Pxn 4通过保护线粒体免受ROS损伤从而保护利什曼原虫的程序性细胞死亡，在寄生虫在宿主中的存活中发挥重要的功能作用。本文的具体目标是：（1）研究L。donovani组织蛋白酶B通过靶向TGF-β在宿主中的存活。(2)确定L.杜氏组织蛋白酶B对利什曼原虫分泌蛋白及其在宿主-寄生虫相互作用中的作用(3)产生FeSODA和Pxn 4无效突变体以及补充寄生虫，并评估其在寄生虫存活和宿主-寄生虫相互作用中的作用。

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.