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: 10197034
• 负责人: Isabelle Coppens
• 金额: $ 20.47万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197034
• 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; Event; Family; Gastrointestinal tract structure; Genes; Genetic; Goals; Histones; Human; Immune; Immune mediated destruction; 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.

概括 弓形虫Gondii是一种机会性的原生动物寄生虫，对艾滋病/HIV患者构成了重大风险。这 目前可用的毒性药物具有明显的不良反应，无法无效地射击长期 生活在大脑和肌肉中的组织囊肿。识别新药物靶标对于控制弓形虫至关重要 新药感染。 T. gondii的自然感染是通过口服摄入组织囊肿（包含）发生的 Bradyzoites）或环境卵囊（含有子孢子）。摄入组织囊肿后，Excysted Bradyzoites入侵肠细胞，它们转化为快速复制的tachyzoite形式。经过几个周期之后 在小肠和肠道器官中的复制，tachyzoites迁移到层次上 血液到达大脑和肌肉以形成组织囊肿。弓形虫囊肿如何能够在 摊位和肠道组织的严峻环境，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），需要用宿主口服摄入 引发感染，具有编码序列蛋白酶抑制剂的基因的共同存在 宿主丝氨酸蛋白。我们的建议重点是高度丰富的丝氨酸蛋白抑制剂的贡献 T. gondii，命名为TGPI-1，以保护寄生虫免受肠腔中存在的宿主串行保护酶 （胰腺弹性蛋白酶，胰蛋白酶，胰胆红蛋白蛋白酶）和/或由固有层（中性粒细胞）中的免疫细胞分泌 弹性酶，组织蛋白酶G）。我们表明，TGPI-1由密集颗粒中的tachyzoites和Bradyzoites分泌， 并在体外抑制胰蛋白酶，胰胆红蛋白蛋白酶和中性粒细胞弹性酶。与野生型寄生虫相比 不足的寄生虫（膀胱源性，II型菌株）更容易受到添加到培养基中的连续蛋白酶的影响 口服组织囊肿后，小鼠胃肠道的传播减少了。我们的 假设是TGPI-1在肠道感染发作时有助于保护弓形虫。具体目标 1将确定TGPI-1靶标和位点，其中ΔTGPI-1寄生虫在胃肠道中被杀死以揭示 TGPI-1被分泌以保护肠道中的弓形虫的地点。具体目标2将重点放在贡献上 TGPI-1通过抑制被激活的中性粒细胞弹性酶来保护椎上层的寄生虫 嗜中性粒细胞或在中性粒细胞外陷阱（NET）延伸过程中释放的中性粒细胞或释放 中性粒细胞弹性蛋白酶高度丰富。从治疗观点来看，TGPI-1可能是新近识别的 病毒因子及其药物抑制与当前药物治疗结合使用，可能会增加 抗氧化剂治疗的功效。

项目成果

MalDA, Accelerating Malaria Drug Discovery.

• DOI: 10.1016/j.pt.2021.01.009
• 发表时间: 2021-06
• 期刊: Trends in parasitology
• 影响因子: 9.6
• 作者: Yang T;Ottilie S;Istvan ES;Godinez-Macias KP;Lukens AK;Baragaña B;Campo B;Walpole C;Niles JC;Chibale K;Dechering KJ;Llinás M;Lee MCS;Kato N;Wyllie S;McNamara CW;Gamo FJ;Burrows J;Fidock DA;Goldberg DE;Gilbert IH;Wirth DF;Winzeler EA;Malaria Drug Accelerator Consortium
• 通讯作者: Malaria Drug Accelerator Consortium