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

胃肠道中的弓形虫：寄生虫蛋白酶抑制剂的保护作用

• 批准号: 10082715
• 负责人: Isabelle Coppens
• 金额: $ 24.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-17 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082715
• 关键词: AIDS/HIV problem; Abbreviations; Adverse effects; Affinity; Affinity Chromatography; Animals; Antibody Affinity; Basement membrane; Binding; Biochemical; Biological Assay; Blood; Blood Circulation; Blood Vessels; Brain; Cathepsin G; Cell Death; Cells; Cessation of life; Chorioretinitis; Code; Complement; Cyst; Cytoplasmic Granules; DNA; Data; Development; Disease; Encephalitis; Enterocytes; Environment; Enzymes; Epithelial; Epithelial Cells; Epithelium; Event; Family; Gastrointestinal tract structure; Genes; Genetic; Goals; Histones; Human; Immune; Immune response; Immunocompetent; Immunocompromised Host; In Vitro; Individual; Infection; Ingestion; Internet; Intestines; Invaded; Lactoferrin; Lamina Propria; Leukocyte Elastase; Leukocyte L1 Antigen Complex; Location; Longevity; Loose connective tissue; Lymphatic; Mass Spectrum Analysis; Membrane; Monitor; Morphology; Muramidase; Mus; Muscle; Myocarditis; Myocardium; NADPH Oxidase; Names; Natural Immunity; Oocysts; Oral; Oral Administration; Oral Ingestion; Organ; Pancreatic Elastase; Parasites; Patients; Peptide Hydrolases; Phagocytosis; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phase; Play; Predisposition; Prevalence; Protease Inhibitor; Proteins; Recombinants; Resistance; Respiratory Burst; Risk; Role; Running; Rupture; Serine Protease; Serine Proteinase Inhibitors; Site; Skeletal Muscle; Small Intestines; Sporozoites; Stomach; Structure; Therapeutic; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Travel; Trypsin; Vaccines; Vacuole; Virulence Factors; alpha-Defensins; antimicrobial; antimicrobial peptide; assault; cell type; chemokine; chymotrypsin; extracellular; in vitro Assay; intraperitoneal; member; monocyte; muscle form; neutrophil; new therapeutic target; novel therapeutics; pathogen; response; scaffold

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.

总结 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。的 目前可获得的用于弓形体病的药物具有显著的副作用， 活组织囊肿位于大脑和肌肉。确定新的药物靶点是控制弓形虫的当务之急 感染新的药物。自然感染T.弓形虫通过口服摄入组织囊肿（包含 缓殖子）或环境卵囊（含有子孢子）。摄入组织囊肿后， 缓殖子侵入肠上皮细胞，在那里它们转变成快速复制的速殖子形式。经过几个周期的 当速殖子在小肠和肠器官中复制时，速殖子迁移到固有层，在那里它们进入肠粘膜。 血液到达大脑和肌肉形成组织囊肿。弓形虫包囊如何能够在 胃和肠道组织的恶劣环境，并逃避固有层中免疫细胞的破坏 仍然是未知的。球虫寄生虫（例如，弓形虫），其需要被宿主口服摄入以 启动感染，共同存在编码丝氨酸蛋白酶抑制剂的基因，其可以靶向 宿主丝氨酸蛋白酶。我们的建议集中在一个高度丰富的丝氨酸蛋白酶抑制剂的贡献， T.弓形虫，命名为TgPI-1，以保护寄生虫免受肠腔中存在的宿主丝氨酸蛋白酶的侵害 （胰弹性蛋白酶、胰蛋白酶、胰凝乳蛋白酶）和/或由固有层中的免疫细胞（嗜中性粒细胞）分泌 弹性蛋白酶、组织蛋白酶G）。我们发现TgPI-1由速殖子和缓殖子从致密颗粒分泌， 并在体外抑制胰蛋白酶、胰凝乳蛋白酶和中性粒细胞弹性蛋白酶。与野生型寄生虫相比， 缺陷型寄生虫（囊源性，II型菌株）更易受加入培养基中的丝氨酸蛋白酶的影响， 在口服给药组织囊肿后，减少了在小鼠胃肠道中的传播。我们 假设TgPI-1有助于保护T。在肠道感染开始时感染弓形虫。具体目标 1将确定TgPI-1靶标和ΔTgPI-1寄生虫在胃肠道中被杀死的部位，以揭示 分泌TgPI-1以保护肠道中的弓形虫的位点。具体目标2将侧重于贡献 的TgPI-1，以保护固有层中的寄生虫，通过抑制中性粒细胞弹性蛋白酶分泌的活化 中性粒细胞或在中性粒细胞胞外陷阱（NET）挤出期间由垂死的中性粒细胞释放，其中 中性粒细胞弹性蛋白酶是高度丰富的。从治疗的角度来看，TgPI-1可能是一种新发现的治疗方法。 毒力因子及其药理学抑制与目前的药物治疗相结合，可能会增加 抗弓形虫治疗的有效性。