Mucosal determinants of Cryptosporidium infection

项目负责人/主要研究者（最后一名、第一名、中间名）：Okhuysen、巴勃罗C/Chappell、Cynthia L 标题：隐孢子虫感染的粘膜清除剂 PI：巴勃罗C. Okhuysen，MD MD安德森癌症中心 主要研究者：Cynthia L. Chappell博士 UT公共卫生学院 项目摘要 隐孢子虫引起腹泻病，在儿童中的流行率仅次于轮状病毒， 发展地区。这种环境适应性强、传染性强的NIAID B类病原体也是导致 在美国爆发的水传播性腹泻。在营养不良的儿童、老年人和免疫抑制者中， 感染会导致慢性衰弱性疾病，甚至致命。寄生虫不能有效地生长 在体外，没有有效的治疗方法。新的方法来研究这种代理的病理生理学是 为了制定治疗和控制的策略，需要。我们之前已经证明，当暴露在 健康的成年志愿者表现出依赖于寄生虫的不同结果， 基因型/物种。这些差异的分子基础是未知的。我们最近还表明，当 暴露于寄生虫，志愿者与高水平的肠道吲哚（IND），肠道细菌的产物， 但对于这种关联存在的原因或其作用机制尚不清楚。因此，在本发明中， 该提案的总体目标是研究所观察到的保护性的潜在机制， 寄生虫基因组多样性的背景下的现象。为此，我们将使用新的技术，如全 长度（FL）基因组测序和类器官培养，以检查微生物组组成的影响， IND化合物（IC）对细胞系和肠道类器官培养物中寄生虫感染性的影响。基因组测序 宿主和/或寄生虫的RNA表达将有助于描述宿主的基本成分- 寄生虫的相互作用，从而提出了潜在的干预措施。具体来说，我们将研究微生物组， 组成和产生FL基因组的粪便样品： 隐孢子虫; HIV感染者;和免疫抑制的癌症患者，后两组与 社区获得性隐孢子虫我们将对寄生虫的基因组进行测序，并测试IC的直接影响。 通过监测寄生虫的结合、入侵和复制，研究寄生虫在细胞系和类器官中的感染性。 我们还将测量受感染细胞产生的细胞因子和其他免疫调节因子的表达 以更好地了解免疫应答在隐孢子虫发病机制中的作用。最后，在证明 概念实验，我们将剥夺成年小鼠的IND，然后用隐孢子虫攻击它们，并监测 感染宏基因组测序将监测细菌群落的变化前后 操纵综上所述，我们希望这些实验将揭示IND中涉及的机制， 相关的保护基因型多样的隐孢子虫感染。我们假设IND和/或 隐孢子虫感染通过芳香烃上调宿主细胞表达和分泌细胞因子 受体途径，参与免疫反应。我们希望这些研究将奠定基础 用于开发新的治疗方式，如IND或IC，或可能使用IND生产细菌作为 益生菌，以增加肠道吲哚水平，从而促进肠道环境，这将使个人少 易感染隐孢子虫病。 OMB编号0925-0001/0002（2018年1月批准至2020年3月31日修订版）页码续页格式页码

Program Director/Principal Investigator (Last, First, Middle): Okhuysen, Pablo C/Chappell, Cynthia L Title: MUCOSAL DETERMINANTS OF CRYPTOSPORIDIUM INFECTION PI: Pablo C. Okhuysen, MD MD Anderson Cancer Center PI (subcontract): Cynthia L. Chappell, PhD UT School of Public Health PROJECT SUMMARY Cryptosporidium causes diarrheal illness and is second only to Rotavirus in prevalence among children in developing areas. This environmentally resilient, highly infectious NIAID category B agent is also a major cause of waterborne outbreaks of diarrhea in the US. In malnourished children, the elderly and in immunosuppressed individuals, infection causes chronic debilitating illness that can be fatal. The parasite cannot be grown efficiently in vitro and no effective treatment is available. New approaches to study the pathophysiology of this agent are needed in order to develop strategies for treatment and control. We have previously shown that when exposed to the parasite, healthy adult volunteers demonstrate distinct outcomes that are dependent on parasite genotype/species. The molecular basis for these differences are unknown. We also recently showed that when exposed to the parasite, volunteers with high levels of intestinal indole (IND), a gut bacterial product, are protected from infection but nothing is known about why this association exists or its mechanism of action. Thus, the overarching goal of this proposal is to examine potential mechanisms for the observed protective phenomenon in the context of parasite genome diversity. To this end, we will use new techniques, such as full length (FL) genome sequencing and organoid cultures, to examine the effect of microbiome composition and IND compounds (ICs) on parasite infectivity in cell lines and intestinal organoid cultures. Genomic sequencing and/or RNA expression of the host and/or parasite will help to delineate essential components of the host- parasite interaction thereby suggesting potential interventions. Specifically, we will study the microbiome composition and generate FL genomes from stool samples of: adults previously challenged with Cryptosporidium; HIV-infected individuals; and immunosuppressed cancer patients, the latter two groups with community-acquired Cryptosporidium. We will sequence parasite genomes and test the direct effects of ICs on the parasite infectivity in cell lines and organoids by monitoring parasite binding, invasion, and replication. We will also measure expression of cytokines and other immunomodulatory factors produced by infected cells to better understand the role of the immune response in Cryptosporidium pathogenesis. Finally, in a proof of concept experiment, we will deprive adult mice of IND, then challenge them with Cryptosporidium, and monitor for infection. Metagenomic sequencing will monitor changes in the bacterial communities before and after manipulation. Taken together, we expect these experiments will shed light on the mechanism involved in IND- associated protection from genotypically diverse Cryptosporidium infections. We postulate that IND and/or Cryptosporidium infection upregulate host cell expression and secretion of cytokines via the aryl hydrocarbon receptor pathway, which is involved in the immune response. We expect these studies will lay the foundation for developing new treatment modalities, such as IND or ICs, or possibly the use of IND-producing bacteria as probiotics to increase gut indole levels, thus promoting a gut environment which would make individuals less susceptible to cryptosporidiosis. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page