Cryptosporidium is a health hazard to people worldwide but is especially problematic in immunocompromised individuals and children. The parasite causes diarrheal illness second only to Rotavirus among children living in developing areas of the world. No effective treatment has been developed for these vulnerable populations, and new approaches to both treatment and control are needed. In 2016, we demonstrated that the presence of high levels of a gut bacterial product, indole, is associated with protection from Cryptosporidium infection. However, nothing is known about how or why this association exists and hypothesized that this correlated with host microbiome diversity. Thus, the purpose of this new study is to examine potential mechanisms for the observed protective phenomenon in the context of genome diversity. 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 cryptosporidiosis.
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 test the direct effects of indole and six biologically-relevant indole metabolites on the parasite and host cells, including transformed human cell lines, enteroids and pulmonoids. Specifically, we will examine the effects of indole and its metabolites on the: (1) viability of isolated oocysts and sporozoites and (2) in vitro infectivity and development of Cryptosporidium in HCT-8 and A549 cells by monitoring parasite binding, invasion, and replication. We will also examine cytokines and other immunomodulatory factors produced by infected cells to shed light on the role of the immune system in relation to indole and its role in Cryptosporidium pathogenesis. We expect these experiments to identify the mechanism involved in indole-associated protection from Cryptosporidium infection.
These studies provide the groundwork for developing new treatment modalities, such as the use of indole and/or its metabolites as drugs or possibly indole-producing bacteria as probiotics to increase gut indole levels, thus promoting a gut environment that would make individuals, especially the immunocompromised, less susceptible to cryptosporidiosis.