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，每年造成50多万新病例和59,000人死亡。利什曼原虫已经进化出许多机制来逃避宿主的免疫反应，并在巨噬细胞内生存。它通过抑制抗原提呈、抑制细胞因子的产生和诱导免疫抑制分子来调节免疫反应。转化生长因子β是利什曼原虫与宿主巨噬细胞相互作用以逃避宿主巨噬细胞杀菌活性过程中产生的主要促炎细胞因子之一。利什曼原虫组织蛋白B半胱氨酸蛋白酶参与了这一过程。我们已经证明利什曼原虫组织蛋白B在体外裂解并激活前转化生长因子-1。然而，组织蛋白酶B在体内对转化生长因子-β的影响还没有研究。我们还证实了组织蛋白酶B基因的干扰导致了杜氏乳杆菌蛋白质组的调节，主要影响了参与氧化还原的分泌蛋白，提示组织蛋白酶B在基于外切体的蛋白质分泌和抗氧化防御系统中发挥作用。利什曼原虫具有铁超氧化物歧化酶(FeSOD)和过氧化物酶，以解毒活性氧物种(ROS)，从而在巨噬细胞内生存。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)外显体分泌的机制尚不清楚。此外，利什曼原虫的抗氧化蛋白FeSODS和过氧化物酶在宿主-寄生虫相互作用中的作用以及抗氧化防御系统的机制还不清楚。我们已经证明，超氧化物歧化酶-A(FeSODA)和过氧化物酶-4(Pxn4)是针对线粒体的，并在体外保护利什曼原虫免受线粒体来源的ROS损伤和细胞程序性死亡。然而，线粒体保护免受ROS损伤的机制尚不清楚，ROS损伤将导致细胞程序性死亡。进一步研究FeSODA和Pxn4在体内存活和程序性细胞死亡中的作用和机制(S)，将为利什曼原虫与宿主相互作用的机制(S)提供更多的见解。因此，了解组织蛋白酶B影响巨噬细胞信号转导的基本机制(S)以及组织蛋白酶B在调节巨噬细胞信号转导中的作用以及FeSOD和Pxn4在利什曼原虫细胞程序性死亡中的作用，将有助于了解宿主与寄生虫的相互作用，并将对微生物使用类似的策略调节巨噬细胞信号转导具有重要意义。**我们假设：*(1)利什曼原虫组织蛋白酶B通过靶向转化生长因子-β而在宿主存活中发挥作用，并在包括抗氧化蛋白在内的利什曼原虫毒力因子的分泌中调节巨噬细胞的信号和功能。*(2)FeSODA和Pxn4都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生存中发挥重要作用。**这项建议的具体目标是：*(1)通过靶向转化生长因子β来研究组织蛋白酶B在宿主生存中的作用。*(2)确定组织蛋白酶B对利什曼原虫分泌蛋白的影响及其在宿主-寄生虫相互作用中的作用。*(3)产生FeSODA和Pxn4缺失突变以及补充寄生虫，并评估它们在寄生虫生存和宿主-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.