PROJECT SUMMARY The inability of Entamoeba histolytica to form infectious cysts in the laboratory setting has greatly hindered investigation of this crucial stage in the infection and disease cycle of this human pathogen that causes amoebic dysentery in ~100 million people each year worldwide. Instead, scientists have been forced to rely on studies with the distantly related reptile pathogen Entamoeba invadens. The long-term goal of our research program is to determine how E. histolytica adapts to different environments it encounters during infection and the disease process. In particular, we are interested in how E. histolytica adapts to the environment of the large intestine in order to colonize there and spread disease by formation and dissemination of infectious cysts. We have now established a reproducible system for encystation and excystation of E. histolytica in culture. This major technological advance enables us to pursue an understanding of how E. histolytica senses and responds to environmental cues that signal conversion from motile trophozoite to infectious cyst and back. As part of our long-term goal, the overall objective of this proposal is to identify and characterize genes responsible for initiation of encystation. The rationale for the proposed project is that understanding how E. histolytica senses and responds to its environment through encystation will lead to a better understanding of how this pathogen can survive and thrive as it encounters very diverse environments during different stages of its infectious cycle. We will pursue two specific aims: (1) identify encystation initiation genes using RNAseq; and (2) screen an overexpression library for genes involved in initiation of encystation in E. histolytica. Candidate genes identified through these two approaches will be validated through analysis of gene silenced and gene overexpression strains. We will evaluate these strains for their ability to encyst, excyst, and establish standard trophozoite growth as well as their responses to other stresses such as heat, oxidative, and nitrosative stress to determine whether any of the candidate genes play a general stress response role. As part of the proposed research, we will optimize our encystation protocol and determine other environmental signals that trigger more rapid encystation. The complementary RNAseq and library screening approaches should allow us to identify genes required for the earliest stages of encystation prior to chitin cell wall formation as well as regulatory genes. The significance of this research is that we can now begin to understand the interplay of environmental signals that regulate encystation and how these signals are acted upon by E. histolytica. This research will have an important impact on the field in that for the first time the processes involved in stage conversion can be fully studied directly in the human pathogen to provide a better understanding of how E. histolytica can thrive during colonization and continue to propagate disease through spread of infectious cysts.

PROJECT NARRATIVE The proposed research is relevant to public health because it investigates a critical segment of the infection cycle of Entamoeba histolytica, which causes an estimated 100 million cases of amoebic dysentery each year worldwide and is a major public health problem in many developing countries. We have developed a method for producing infectious cysts in the laboratory and can now capitalize on this major advancement to study how E. histolytica senses and responds to environmental signals to form cysts to continue the cycle of infection. This understanding provides a critical foundation for developing mechanisms targeting this stage of infection as a way to reduce the spread of this disease pursuant to part of the NIH’s mission.