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。目前还没有针对隐孢子虫的人类疫苗，用于治疗隐孢子虫的主要药物对免疫抑制的人无效。这个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育是否是维持隐孢子虫慢性感染所必需的。简言之，卵囊含有四个单倍体子孢子，其感染上皮细胞并作为裂殖子无性繁殖。裂殖子分化为有性形式，有性形式融合形成二倍体合子。受精卵形成卵囊壁，在卵囊壁内发生减数分裂并发育出四个子孢子。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。 这里的创新和风险是结合联合收割机的卵囊壁，这是我们实验室的专业，与最近开发的方法遗传操纵隐孢子虫在小鼠和无限期地保持整个隐孢子虫的生命周期在中空纤维培养的HCT-8细胞的生化和形态学研究。在中空纤维培养物中遗传操作隐孢子虫的优点是1）获得的卵囊没有粪便细菌的污染，以及2）避免了将电穿孔的滋养体植入小鼠肠中的存活手术。此外，我们提出用野生型寄生虫共感染敲除，以1）使二倍体合子可用于鉴定卵囊壁合成和/或孢子形成的必需基因，和2）产生敲除这些必需基因的子孢子。 在第一个特定目标中，我们将敲除参与卵囊壁形成的基因（例如，在壁内层形成原纤维的木聚糖合酶或产生抗酸脂质的聚酮合酶），并确定对卵囊壁形成和子孢子产生的影响。我们还将敲除减数分裂相关基因，减数分裂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于卵囊壁和子孢子的发育是必不可少的，如果自身感染被阻断，则提示新的药物靶点以预防慢性感染。 在第二个特定目标中，我们将用敲除子孢子和野生型对照感染小鼠组，并使用非侵入性体外成像系统（IVIS）监测感染。如果基因敲除阻止卵囊壁形成和孢子形成，我们预计自身感染将被阻断，并且与报告细胞系相比，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.