Hypothesis: Activation of human K+ channels during amebic trogocytosis is the causal mediator of cell death. Preliminary data: K+ channels were identified in a novel forward genetic screen for host factors required for trogocytic killing by Entamoeba histolytica. Key preliminary experimental data also support the importance of K+ channels including: (1) E. histolytica activated K+ channels prior to killing target cells; and (2) inhibition of K+ channels protected cells from amebic killing. Approach: We will extend this discovery by analyzing specific colonic K+ channels as potential drug targets in the human enteroid model of amebiasis. We will test CRISPR-Cas9 deletions of high confidence candidate K+ channels genes in human enteroids. Gene deletion studies will be paired with pharmacologic inhibitor testing in enteroids with the goal of providing proof-of-principle for K+ channel inhibitrs as novel therapeutics. Successful completion of these studies will determine if human K+ channels are novel host-directed, druggable targets for amebiasis. Significance: E. histolytica is a diarrheal disease with significant global morbidity and mortality, especially in children in the developing world. There is a single class of drugs for invasive disease and toxicity and the emergence of resistance are clinical concerns. The highly pathogenic free-living amebae (Naegleria fowleri, Acanthamoeba sp., and Balamuthia mandrillaris) also kill human cells via trogocytosis, therefore our studies aiming to prevent trogocytic death in enteric amebiasis, may also offer therapy for the free-living amebae. Innovative aspects of the proposal are: the forward genetic screen that identified human K+ channels as a potential host therapeutic target; the focus on amebic trogocytic killing, a unique cellular process shared by parasitic E. histolytica and the free-living amebae; and by the use of cutting edge technology (a human colonic enteroid model and validated CRISPR gene deletions). The environment for the work is Dr. William A. Petri's laboratory at The University of Virginia. Dr. Petri has been a leader in the fied of amebiasis research for 25 years and his laboratory is an intellectually exciting and rigorous environment for the study of enteric infections. There are 7 postdoctoral fellows and 5 graduate students, with independent yet complementary projects. The laboratory contains all the necessary resources and expertise to accomplish high-impact research and great emphasis is placed on collaboration and sharing of ideas and techniques, as well as independence and career development for the fellows and graduate students. Weekly laboratory meetings and daily informal discussions (facilitated by the presence of Dr. Petri's office within the laboratory serve to promote the ongoing research program.
Using a forward genetic screen we discovered that Entamoeba histolytica activates human potassium (K+) channels to kill cells by amebic trogocytosis. We found that knockdown of K+ channels made cells resistant to E. histolytica cell killing. To understand how parasites exploit human K+ channel activation to invade the colonic epithelium we will: 1) Identify the colonic K+ channel activated by E. histolytica in colonic invasion of human enteroids; and 2) test the efficacy of pharmacologic K+ channel inhibitors as potential virulence-blocking drugs in the innovative enteroid model of amebiasis. Successful completion of these aims has the potential to define a novel therapeutic target for an important parasitic disease.
