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

• 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
• 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

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.

隐孢子虫导致婴儿严重腹泻和免疫抑制者慢性腹泻。目前还没有针对这种寄生虫的人类疫苗，用于治疗隐孢子虫的主要药物对免疫抑制的人无效。R21建议的目的是确定卵囊壁的合成和/或感染性子孢子的发育是否对于维持隐孢子虫的慢性感染是必要的。简单地说，卵囊包含四个单倍体的子孢子，它们感染上皮细胞并以裂殖子的形式无性繁殖。裂殖子分化为有性形式，融合形成二倍体受精卵。然后受精卵形成卵囊壁，在卵囊壁内发生减数分裂，发育出四个子孢子。由于隐孢子虫的无性繁殖是有时间限制的，卵囊的自体感染似乎是维持严重或慢性感染所必需的。这里的创新和风险是将对卵囊壁的生化和形态研究与最近开发的方法结合起来，在小鼠中对隐孢子虫进行遗传操作，并在HCT-8细胞的中空纤维培养中无限期地维持整个隐孢子虫的生命周期。在中空纤维培养中对隐孢子虫进行基因操作的优点是：1)获得的卵囊不受粪便细菌的污染；2)避免了将电穿孔滋养体植入小鼠肠道的存活手术。此外，我们建议将敲除基因与野生型寄生虫混合感染，以1)使二倍体受精卵用于鉴定卵囊壁合成和/或产孢子所必需的基因，以及2)产生这些必需基因被敲除的子孢子。在第一个具体目标中，我们将敲除与卵囊壁形成有关的基因(例如，在卵囊壁内层形成纤维的木聚糖合成酶或产生抗酸脂肪的聚酮合酶)，并确定在中空纤维培养中对卵囊壁形成和子孢子产生的影响。我们还将敲除涉及减数分裂的基因，这是在卵囊壁内产生四个感染性子孢子的第一步。基因敲除将告诉我们哪些基因对卵囊壁和子孢子的发育至关重要，如果自身感染被阻止，还将提出预防慢性感染的新药物靶点。在第二个特定目标中，我们将用敲除的子孢子和野生型对照感染多组小鼠，并使用非侵入性体外成像系统(IVIS)监测感染情况。如果基因敲除阻止卵囊壁的形成和孢子形成，我们预计自体感染将被阻止，与报道线相比，感染将是有时间限制的。我们还将比较基因操作的卵囊和子孢子对人类3D肠道细胞组织的感染。