Mucosal determinants of Cryptosporidium infection

项目主任/首席调查员(最后、第一、中间)：奥库伊森、巴勃罗·C/查佩尔、辛西娅·L 隐孢子虫感染的粘膜决定因素 少年派：巴勃罗·C·奥凯森，医学博士 安德森癌症中心 PI(分包合同)：辛西娅·L·查佩尔，博士 德克萨斯大学公共卫生学院 项目总结 隐孢子虫引起腹泻疾病，在儿童中的流行率仅次于轮状病毒。 发展中地区。这种环境适应性强、传染性强的B类NIAID病毒也是一个主要原因 在美国爆发的水源性腹泻。在营养不良的儿童、老年人和免疫抑制患者中 就个人而言，感染会导致慢性衰弱疾病，可能是致命的。这种寄生虫不能高效生长。 在体外，没有有效的治疗方法。研究这种制剂的病理生理学的新方法是 为制定治疗和控制战略而需要的。我们之前已经证明，当被曝光时 对于寄生虫来说，健康的成年志愿者表现出依赖于寄生虫的不同结果。 基因型别/物种。这些差异的分子基础尚不清楚。我们最近还表明，当 暴露在寄生虫中，肠道细菌产物--肠道吲哚(IND)水平较高的志愿者 保护免受感染，但对这种联系为什么存在或其作用机制一无所知。因此， 这项建议的首要目标是检查观察到的保护的潜在机制 寄生虫基因组多样性背景下的现象。为此，我们将使用新技术，例如Full 长度(FL)基因组测序和有机物培养，以检查微生物组组成和 Ind化合物(IC)对细胞系和肠道器官培养中寄生虫感染性的影响。基因组测序 和/或寄主和/或寄生虫的RNA表达将有助于描绘寄主的基本成分- 寄生虫的相互作用，从而暗示了潜在的干预。具体地说，我们将研究微生物组 从粪便样本中组成并生成FL基因组：以前挑战过的成年人 隐孢子虫；艾滋病毒感染者；以及免疫抑制的癌症患者，后两组 社区获得性隐孢子虫。我们将对寄生虫基因组进行测序，并测试IC的直接影响 通过监测寄生虫的结合、入侵和复制来研究寄生虫在细胞系和器官中的感染性。 我们还将检测感染细胞产生的细胞因子和其他免疫调节因子的表达。 为了更好地了解免疫反应在隐孢子虫发病机制中的作用。最后，为了证明 概念实验，我们将剥夺成年小鼠的IND，然后用隐孢子虫挑战它们，并监测 用来治疗感染。元基因组测序将监测细菌群落在前后的变化 操纵。综上所述，我们预计这些实验将揭示IND-1的相关机制。 对基因多样性隐孢子虫感染的相关保护。我们假定IND和/或 隐孢子虫感染通过芳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为我们奠定基础 用于开发新的治疗模式，如IND或ICS，或可能使用产生IND的细菌作为 益生菌提高肠道吲哚水平，从而促进肠道环境，使个体减少 易患隐孢子虫病的。 OMB编号0925-0001/0002(01/18修订版批准至2020年3月31日)页面续格式页面

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