Genetic modification of cultured Cryptosporidium to test the autoinfection model

对培养的隐孢子虫进行基因改造以测试自身感染模型

基本信息

批准号：
9305341
负责人：
John C. Samuelson
金额：
$ 24.68万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9305341
关键词：
Acid Fast Bacillae Staining Method Asexual Reproduction Bacteria Biochemical Cells Chronic Chronic Disease Cryptosporidium Cyst Development Diarrhea Diploidy Drug usage Electrons Entamoeba Epithelial Cells Essential Genes Exposure to Feces Fiber Gene Targeting Genes Genetic Giardia Glycoproteins Goals Haploidy Human Implant In Vitro Infant Infection Interruption Intestines Knock-out Life Cycle Stages Lipids Longitudinal Studies Luciferases Meiosis Methods Modeling Modification Monitor Morphology Mus Mutation Mycoses Oocysts Operative Surgical Procedures Oral Parasites Persons Population Production Protein Biosynthesis Proteins Protozoa Reporter Risk Route Sporozoites System Testing Thick Thinness Time Tissue Model Tissues Toxoplasma Vaccines Xylans asexual excystation glucan synthase imaging system immunosuppressed inhibitor/antagonist innovation knockout gene medical specialties mouse model new therapeutic target polyketide synthase prevent transmission process zygote

Acid Fast Bacillae Staining Method Asexual Reproduction Bacteria Biochemical Cells Chronic Chronic Disease Cryptosporidium Cyst Development Diarrhea Diploidy Drug usage Electrons Entamoeba Epithelial Cells Essential Genes Exposure to Feces Fiber Gene Targeting Genes Genetic Giardia Glycoproteins Goals Haploidy Human Implant In Vitro Infant Infection Interruption Intestines Knock-out Life Cycle Stages Lipids Longitudinal Studies Luciferases Meiosis Methods Modeling Modification Monitor Morphology Mus Mutation Mycoses Oocysts Operative Surgical Procedures Oral Parasites Persons Population Production Protein Biosynthesis Proteins Protozoa Reporter Risk Route Sporozoites System Testing Thick Thinness Time Tissue Model Tissues Toxoplasma Vaccines Xylans asexual excystation glucan synthase imaging system immunosuppressed inhibitor/antagonist innovation knockout gene medical specialties mouse model new therapeutic target polyketide synthase prevent transmission process zygote

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项目摘要

Cryptosporidium causes severe diarrhea in infants and chronic diarrhea in immunosuppressed persons. There are no human vaccines against the parasite, and the primary drug used to treat Cryptosporidium is not effective in immunosuppressed persons. The goal of this R21 proposal is to determine whether synthesis of the oocyst wall and/or development of infectious sporozoites are necessary for maintaining chronic infections by Cryptosporidium. Briefly, oocysts contain four haploid sporozoites, which infect epithelial cells and reproduce asexually as merozoites. Merozoites differentiate into sexual forms, which fuse to form diploid zygotes. Zygotes then make the oocyst wall, within which meiosis occurs and four sporozoites develop. Because asexual reproduction by Cryptosporidium is time-limited, autoinfection by oocysts appears to be necessary for maintaining severe or chronic infections. The innovation and risk here is to combine biochemical and morphological studies of the oocyst wall, which is a specialty of our lab, with recently developed methods for genetically manipulating Cryptosporidium in mice and for maintaining indefinitely the entire Cryptosporidium life cycle in hollow fiber cultures of HCT-8 cells. The advantages of genetically manipulating Cryptosporidium in hollow fiber cultures are 1) oocysts are obtained without contamination by stool bacteria and 2) survival surgeries to implant electroporated trophozoites into the mouse intestine are avoided. In addition, we propose to co-infect knockouts with wild-type parasites to 1) make diploid zygotes useful for identification of essential genes for oocyst wall synthesis and/or sporulation and 2) produce sporozoites with these essential genes knocked out. In the first Specific Aim we will knock out genes involved in oocyst wall formation (e.g. xylan synthase that forms fibrils in the inner layer of the wall or polyketide synthase that makes acid-fast lipids) and determine the effects on oocyst wall formation and sporozoite production in hollow fiber cultures. We will also knock out genes involved in meiosis, which is the first step to produce four infectious sporozoites within oocyst walls. The knockouts will tell us what genes are essential for development of oocyst walls and sporozoites and, if autoinfection is blocked, suggest novel drug targets to prevent chronic infection. In the second Specific Aim we will infect sets of mice with knockout sporozoites and wild-type controls and monitor the infections using non-invasive in vitro imaging system (IVIS). If the gene knockouts prevent oocyst wall formation and sporulation, we expect that autoinfection will be blocked, and infections will be time-limited compared to the reporter line. We will also compare infection of human 3D intestinal cell tissues by genetically manipulated oocysts and sporozoites.

隐孢子虫在免疫抑制人群中引起婴儿和慢性腹泻的严重腹泻。没有针对寄生虫的人类疫苗，用于治疗隐孢子虫的主要药物在免疫抑制者中无效。该R21提案的目的是确定卵囊壁的合成和/或感染性子孢子的发展是否对于维持隐孢子虫的慢性感染是必要的。简而言之，卵囊含有四个单倍孢子菌，它们会感染上皮细胞并无性繁殖为梅罗寄生虫。 Merozoites differentiate into sexual forms, which fuse to form diploid zygotes.然后，Zygotes成为卵囊壁发生，并在其中发生减数分裂，并发展四个孢子菌。由于隐孢子虫的无性繁殖是时间限制的，因此卵囊自身感染似乎对于维持严重或慢性感染是必要的。这里的创新和风险是结合卵囊壁的生化和形态学研究，这是我们实验室的特殊性，最近开发的方法是通过遗传操纵小鼠中的隐孢子虫和无限期地维持整个HCT-8细胞中空心生命周期的隐孢子虫生命周期的方法。在空心纤维培养物中遗传操纵隐孢子虫的优势是1）卵囊可通过粪便细菌污染而没有污染，而2）避免植入小鼠肠中的植入型电穿孔的滋养器。此外，我们建议将野生型寄生虫共同敲除1）使二倍体合子可用于鉴定卵囊壁合成和/或孢子形成必需基因，以及2）用这些基本基因敲出的基本基因产生孢子虫。在第一个特定目的中，我们将淘汰涉及卵囊壁形成的基因（例如，在壁或聚酮化合物合酶的内层形成纤维的木聚糖合酶，使脂肪脂脂脂质产生），并确定对卵母细胞壁形成和空心纤维培养物中的卵母细胞壁形成和孢子产生的影响。 We will also knock out genes involved in meiosis, which is the first step to produce four infectious sporozoites within oocyst walls.淘汰赛将告诉我们哪些基因对于卵囊壁和孢子菌的发展至关重要，如果自身感染被阻断，请提出新的药物靶标，以防止慢性感染。在第二个特定目的中，我们将用敲除孢子和野生型对照感染一组小鼠，并使用非侵入性体外成像系统（IVIS）监测感染。如果基因敲除可以防止卵囊壁的形成和孢子形成，我们希望自身感染将被阻塞，并且与记者线相比，感染将被限制。我们还将通过遗传操纵的卵囊和孢子岩来比较人类3D肠细胞组织的感染。