The proposed training plan combines intensive didactic and basic laboratory training to prepare Christopher Huston, M.D., for a career investigating host-pathogen biology during invasive amebiasis. Entamoeba histolytica, the protozoan cause of amebic colitis and liver abscess, infects an estimated 50 million people causing up to 100 thousand deaths annually. Amebiasis is an indolent infection and inflammation is sparse given the degree of tissue destruction. Apoptotic host cell killing and phagocytosis further characterize E. histolytica infection. Indeed, clinicians use erythrophagocytosis to distinguish E. histolytica from the non-pathogenic ameba Entamoeba dispar. This project's hypothesis is that apoptotic cell killing followed by rapid phagocytosis limits spillage of toxic intracellular contents and prevents inflammation in an analogous fashion to phagocytosis of apoptotic cells in multicellular organisms. In this way, apoptotic killing and phagocytosis are hypothesized to enable E. histolytica to evade the host immune response and cause disease. Preliminary studies by Dr. Huston have demonstrated apoptotic killing of host cells by E. histolytica and shown that E. histolytica ingested apoptotic cells more efficiently than healthy cells.
Specific Aim 1 is to define the nature and mechanism of host cell surface changes that trigger amebic ingestion using confocal microscopy and flow cytometry to assay phagocytosis.
Specific Aim 2 is to characterize an amebic ABC transporter, EhABC A1, with homology to the phagocytosis-associated transporter Ced-7 from Caenorhabditis elegans. EhABC A1 function/expression will be disrupted using dominant negative mutants, anti-sense RNA expression, and anti-sense peptide nucleic acid oligomers. Results from Specific Aim 1 will clarify the mechanism of amebic cytotoxicity as well as help to identify the amebic phagocytosis receptor.
In Specific Aim 2, successful interference with EhABC A1 to test its role in phagocytosis will help to define the amebic phagocytosis machinery and may provide phagocytosis deficient amoebae to delineate the role of phagocytosis for in vivo virulence.
