Transmembrane Kinases of Entamoeba Histolytica

Buss, Sarah N., Department of Microbiology, University of Virginia
Petri, William, Department of Microbiology, University of Virginia
Brautigan, David, Department of Microbiology, University of Virginia
Amy, Department of Microbiology, University of Virginia
Schafer, Dorothy, Department of Biology, University of Virginia
Ravichandran, Kodi, Department of Microbiology, University of Virginia

The intestinal pathogen, Entamoeba histolytica, is a killer cell that can persist for months within its human host. To survive within the complex intestinal microenvironment, control invasive behavior and evade the immune system, trophozoites must posses a profound ability to sense and respond to environmental stimuli. Interestingly, the E. histolytica genome contains a large family of transmembrane kinases (TMKs) with proposed roles in both amebic response to the environment and immune evasion through antigenic variation. Here, I discount the notion of antigenic variation and instead provide evidence that single cells express multiple TMKs. Laser-capture microdissection and microarray analysis demonstrated that single trophozoites contained multiple TMK transcripts, and antibodies specific for TMK39, TMK54 and PaTMK each homogeneously labeled over 950f cells within a population, indicating that multiple TMKs are expressed at the protein level. TMK39 and TMK54 were then shown to possess non-redundant functions. Confocal microscopy identified different patterns of cell surface expression for the proteins and multispectral imaging flow cytometry demonstrated that TMK39, but not TMK54, co-localized with ingested beads. Moreover, expression of truncated TMK39 interfered with phagocytosis of apoptotic lymphocytes, whereas expression of truncated TMK54 inhibited growth of amoebae and altered surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. To complement our basic research studies, we also addressed an applied research question. In collaboration with the medical diagnostics company Techlab Inc., we developed and evaluated a battery of practical and cost effective diagnostic devices for detection of the NIAID Category B priority pathogens Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica. The tests described herein utilize technologies iii appropriate for the developing world where E. histolytica is prevalent and comprise more rapid and efficient means of detecting the enteric parasites than currently available commercial assays.

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PHD (Doctor of Philosophy)
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