Toxoplasma in the GI tract: Protective role of a parasite protease inhibitor

摘要 弓形虫是一种机会性原生动物寄生虫，对艾滋病/艾滋病毒患者构成重大风险。这个 目前治疗弓形虫病的药物有显著的不良反应，对根除长期感染无效。 位于大脑和肌肉中的活组织囊肿。寻找新的药物靶点是控制弓形虫的当务之急 新药感染。弓形虫的自然感染是通过口服组织包囊(包括 缓殖子)或环境卵囊(包含子孢子)。摄取组织囊肿后，排除 慢殖子侵入肠细胞，在那里它们转变为快速复制的速殖子形式。在经历了几个周期之后 在小肠和肠道器官的复制中，速殖子迁移到固有层，在那里它们进入 血流到达大脑和肌肉，形成组织囊。弓形虫包囊是如何在 胃和肠道组织的恶劣环境，逃脱固有层免疫细胞的破坏 在很大程度上仍不为人所知。球虫寄生虫(例如弓形虫)，需要由宿主口服以 引发感染，共同存在编码丝氨酸蛋白酶抑制物的基因，这些基因可能以 宿主丝氨酸蛋白酶。我们的建议集中在一种高度丰富的丝氨酸蛋白酶抑制剂的贡献 弓形虫，命名为TgPI-1，以保护寄生虫免受肠道中存在的宿主丝氨酸蛋白酶的影响 (胰腺弹性蛋白酶、胰酶、胰凝乳素)和/或由固有层中的免疫细胞(中性粒细胞)分泌 弹性蛋白酶、组织蛋白酶G)。我们发现TgPI-1是由速殖子和缓殖子从致密的颗粒中分泌出来的， 并在体外抑制胰酶、凝乳酶和中性粒细胞弹性蛋白酶。与野生型寄生虫相比，TgPI-1- 缺陷寄生虫(生囊型，II型菌株)更容易受到添加到培养基中的丝氨酸蛋白酶的影响 能减少口服组织囊肿后在小鼠胃肠道内的扩散。我们的 假设TgPI-1在肠道感染时有助于保护弓形虫。特定目标 1将确定TgPI-1的靶点和ΔTgPI-1寄生虫在胃肠道中被杀死的位置，以揭示 分泌TgPI-1以保护肠道内弓形虫的部位。具体目标2将侧重于贡献 TgPI-1通过抑制活化的中性粒细胞弹性蛋白酶分泌保护固有层寄生虫 在中性粒细胞胞外捕捉器(NET)的挤出过程中，中性粒细胞或由垂死的中性粒细胞释放，其中 中性粒细胞弹性蛋白酶含量非常丰富。从治疗的角度来看，TgPI-1可能是一种新发现的 毒力因子及其药理抑制与目前的药物治疗相结合，可能会增加 抗弓形虫治疗的疗效。

SUMMARY Toxoplasma gondii is an opportunistic protozoan parasite that poses a significant risk to AIDS/HIV patients. The currently available drugs for toxoplasmosis have significant adverse effects and are ineffective to eradicate long- lived tissue cysts located in the brain and muscles. Identifying new drug targets is imperative to control Toxoplasma infections with new drugs. Natural infection by T. gondii occurs via oral ingestion of tissue cysts (containing bradyzoites) or environmental oocysts (containing sporozoites). After ingestion of tissue cysts, excysted bradyzoites invade enterocytes where they transform into fast-replicating tachyzoite forms. After several cycles of replication in the small intestine and gut organs, tachyzoites migrate to the lamina propria where they enter the bloodstream to reach the brain and muscles to form tissue cysts. How Toxoplasma cysts are able to survive in the harsh environment of the stomach and gut tissues, and escape destruction by immune cells in the lamina propria remains largely unknown. Coccidian parasites (e.g., Toxoplasma), which need to be ingested orally by a host to initiate an infection, have in common the presence of genes coding for serine protease inhibitors that may target host serine proteases. Our proposal focuses on the contribution of a highly abundant serine protease inhibitor of T. gondii, named TgPI-1 to the protection of the parasite against host serine proteases present in the gut lumen (pancreatic elastase, trypsin, chymotrypsin), and/or secreted by immune cells in the lamina propria (neutrophil elastase, cathepsin G). We showed that TgPI-1 is secreted by tachyzoites and bradyzoites from dense granules, and inhibits trypsin, chymotrypsin and neutrophil elastase in vitro. Compared to wild-type parasites, TgPI-1- deficient parasites (cystogenic, type II strain) are more vulnerable to serine proteases added to the medium and have reduced dissemination in the gastro-intestinal tract of mice after oral administration of tissue cysts. Our hypothesis is that TgPI-1 contributes to the protection of T. gondii at the onset of infection in the gut. Specific Aim 1 will identify the TgPI-1 targets and sites where ΔTgPI-1 parasites are killed in the gastro-intestinal tract to reveal the sites where TgPI-1 is secreted to protect Toxoplasma in the gut. Specific Aim 2 will focus on the contribution of TgPI-1 to protect the parasite in the lamina propria by inhibiting neutrophil elastase secreted by activated neutrophils or released by dying neutrophils during extrusion of Neutrophil Extracellular Traps (NET) wherein neutrophil elastase is highly abundant. From a therapeutic point-of-view, TgPI-1 could be a newly identified virulence factor, and its pharmacological inhibition in combination with current drug treatments, may increase the efficacy of anti-Toxoplasma treatments.