In vitro cultivation and genome sequencing/annotation of Enterocytozoon bieneusi

About This Grant

Abstract Enterocytozoon bieneusi (Eb), an intracellular spore-forming microsporidian fungus, represents the most clinically significant opportunistic infection associated with persistent diarrhea and wasting, occurring in up to 30%–50% of individuals with HIV/AIDS and in immunosuppressed cancer and organ transplant patients. Chronic infections in humans and rhesus macaques share remarkable similarities, primarily localized in the upper small intestine, gallbladder, and bile ducts. The Tufts group has previously pioneered methods for purifying Eb from infected humans and macaques, facilitating genomic surveys, diagnostic antibody reagents’ development, and successful propagation of Eb in immunodeficient rodents. The group has additionally a well-established hollowfiber cultivation system. The U. of Maryland group has an impressive set up of various in vitro models of human intestinal and gallbladder mucosa. The two teams have a long record of collaboration and are eager to harness these resources to advance our research on this critical pathogen. Previous attempts by few investigators to continuously cultivate Eb in cell culture monolayers have been unsuccessful. As mentioned above, our team has the knowledge and hands-on skills to deploy several new in vitro cultivation techniques. We have assembled a unique collection of human specimens, including (i) stem cells isolated from the terminal ileum and gallbladder, (ii) their ex vivo explants counterparts, and (iii) a continuous supply of well characterized source of Eb spores of macaques and human sources, all of which will play an integral role in our proposed research. R61 Phase which includes 3 Specific Aims will develop and optimize in vitro culture systems for long-term continuous propagation of Eb, deploying a) human-derived organoid of polarized epithelial cells, b) organotypic model of the human intestinal mucosa using a rotating-wall-vessel bioreactor and c) separately, evaluating the hollowfiber bioreactor system. R33 Phase which include 3 Specific Aims will confirm and validate the leading system developed under R66 and will in addition complete the sequencing of the Eb genome to improve annotation supporting future genetic research and analyze the transcriptome of in vitro infected cells. In conclusion, cell culture systems of continuous Eb growth and genome sequencing will in future facilitate developing techniques for genetic manipulation of Eb to understand Eb's biology, pathogenicity and host- pathogen interactions, and development of safe and effective drugs to treat Eb infections.