UC Davis

Entamoeba histolytica is a pathogen of global importance, and is responsible for the diarrheal disease amoebiasis. Infection is characterized by massive tissue damage, which is poorly understood and thought to be driven by the contact-dependent cell-killing activity of E. histolytica. E. histolytica performs two main forms of ingestion of human cells: trogocytosis and phagocytosis. Trogocytosis involves taking bites of live host cells and contributes to host cell killing, whereas phagocytosis involves swallowing dead host cells whole. In this body of work, we investigated this distinctions between phagocytosis and trogocytosis, and developed several genetic tools to enable greater study of this parasite. We investigated the connection between host cell attachment and ingestion type by knocking down proteins implicated in attachment using trigger-induced RNA interference. In this process we found that trigger-silenced mutants should be analyzed immediately before phenotypes are lost. We demonstrated for the first time that amoebae acquire and display host cell membrane proteins through trogocytosis but not phagocytosis, allowing them to survive in human serum. To test how host cell deformability influences the type of ingestion performed by amoebae, we designed and optimized an ingestion assay capable of distinguishing between whole host cells, membrane bites, and external host cells. Using this assay, we found that host cells with cytoskeletal proteins knocked down were trogocytosed less by amoebae. We provided practical instruction on how to grow and genetically manipulate E. histolytica in a laboratory research environment. We also constructed the first E. histolytica RNAi knockdown mutant library, and used it to identify and characterize novel genes involved in growth. Finally, we made great progress in establishing CRISPR interference in E. histolytica, discovering that increasing antibiotic concentration affects gene expression, and that a common expression promoter is insufficient for expression of dCas9. Overall, this body of work represents a significant contribution to our understanding of the pathogenesis and cell biology of E. histolytica.