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。内脏利什曼病的病原利什曼原虫复合体每年造成50多万新病例和5.9万例死亡。利什曼原虫已经进化出多种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 （TGF-ß）是利什曼原虫与宿主巨噬细胞相互作用时产生的主要促炎细胞因子之一，以逃避宿主巨噬细胞的杀微生物活性。利什曼原虫组织蛋白酶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酶B在体外切割和激活前tgf -ß1。然而，组织蛋白酶B对TGF-ß在体内的作用尚未见研究。我们还证明，组织蛋白酶B基因的破坏导致L. donovani蛋白质组的调节，主要影响参与氧化还原的分泌蛋白，这表明组织蛋白酶B在基于外泌体的蛋白质分泌和抗氧化防御系统中起作用。利什曼原虫具有铁超氧化物歧化酶（FeSODs）和过氧化物酶来解毒活性氧（ROS），使其在巨噬细胞内存活。组织蛋白酶B影响外泌体分泌利什曼毒力因子（包括参与抗氧化防御系统的蛋白质）的机制尚不清楚。此外，利什曼原虫抗氧化蛋白FeSODs和过氧化物素在宿主-寄生虫相互作用中存活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）和过氧化物毒素-4 （Pxn4）靶向线粒体，并保护体外利什曼原虫免受线粒体来源的ROS损伤和程序性细胞死亡。然而，线粒体免受活性氧损伤的机制尚不清楚，活性氧损伤会导致程序性细胞死亡。进一步研究FeSODA和Pxn4在体内存活和细胞程序性死亡中的作用和机制，将有助于进一步了解利什曼-宿主相互作用的机制。因此，了解组织蛋白酶B在调节巨噬细胞信号传导过程中影响TGF- β和基于外泌体的利什曼原虫蛋白分泌的基本机制，以及FeSOD和Pxn4在利什曼原虫程序性细胞死亡中的作用，将有助于阐明宿主-寄生虫相互作用，并对微生物使用类似策略调节巨噬细胞信号传导具有启示意义。

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.