Genetic modification of cultured Cryptosporidium to test the autoinfection model

隐孢子虫引起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。本R21提案的目的是确定卵囊壁的合成和/或感染性孢子虫的发育是否对维持隐孢子虫的慢性感染是必要的。简单地说，卵囊含有四个单倍体孢子子，它们感染上皮细胞并以分裂子的形式无性繁殖。裂殖子分化为有性形式，融合形成二倍体合子。然后受精卵形成卵囊壁，在卵囊壁内发生减数分裂并发育四个孢子子。由于隐孢子虫的无性繁殖是有时间限制的，卵囊的自身感染似乎是维持严重或慢性感染所必需的。这里的创新和风险在于将我们实验室的专长卵囊壁的生化和形态学研究与最近开发的方法结合起来，这些方法可以在小鼠体内遗传操纵隐孢子虫，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)获得的卵囊不会受到粪便细菌的污染；2)避免了将电穿孔滋养体植入小鼠肠道的生存手术。此外，我们建议将敲除基因与野生型寄生虫共感染，1)使二倍体受精卵用于鉴定卵囊壁合成和/或产孢的必要基因，2)产生敲除这些必要基因的孢子子。在第一个特定目标中，我们将敲除与卵囊壁形成有关的基因（例如，在卵囊壁内层形成原纤维的木聚糖合成酶或产生抗酸脂质的聚酮合成酶），并确定对中空纤维培养中卵囊壁形成和孢子子产生的影响。我们还将敲除与减数分裂有关的基因，这是在卵囊壁内产生四个传染性孢子子的第一步。这些基因敲除将告诉我们哪些基因对卵囊壁和孢子子的发育至关重要，如果自身感染被阻断，我们将提出新的药物靶点来预防慢性感染。在第二个特定目标中，我们将用敲除孢子子和野生型对照感染几组小鼠，并使用无创体外成像系统（IVIS）监测感染情况。如果基因敲除阻止卵囊壁形成和产孢，我们预计自身感染将被阻断，与报告线相比，感染将有时间限制。我们还将比较基因操纵的卵囊和孢子体对人类3D肠细胞组织的感染。

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.