Vermont Center for Immunobiology and Infectious Disease

Research Lab of Christopher Huston, M.D.

Overview

Areas of interest: immunobiology, signal transduction and cell signaling, microbial pathogenesis, Entamoeba histolytica phagocytosis, inflammation.

Entamoeba histolytica is the intestinal protozoan that causes amebic dysentery. Dr. Huston's laboratory is focused on understanding how E. histolytica kills and phagocytoses host cells, two phenotypes correlated with virulence. His studies have shown that E. histolytica induces host cell apoptosis using a contact-dependent mechanism, and ingests host cells only after killing them. The main focus of Dr. Huston's laboratory is now to identify the E. histolytica phagocytosis receptors and the host cell ligands that they recognize. A variety of cell biological and biochemical approaches are being used, including flow cytometry, fluorescent microscopy for imaging both fixed and living parasites, and affinity-based and cross-linking methods for receptor purification. A newly funded project in the lab focuses on identifying human proteins required by the intracellular parasite Cryptosporidium parvum to infect and replicate efficiently within gastrointestinal epithelial cells.

Programs & Projects

In our laboratory we study two waterborne intestinal protozoa that infect humans: Entamoeba histolytica and Cryptosporidium parvum. Both of these parasites are major public health problems in the developing world. They predominantly affect young children and, in the case of Cryptosporidium, immunocompromised individuals such as those with AIDS. Recently, both have garnered increasing interest in the United States due to concern that they could be intentionally introduced into the water supply in an act of bioterrorism.

Entamoeba histolytica, the cause of amebic dysentery and liver abscess, infects an estimated 50 million people annually. The ability of E. histolytica to kill and phagocytose host cells correlates with parasite virulence. In fact, phagocytosis of host erythrocytes is the only feature on light microscopy that distinguishes E. histolytica from the non-pathogenic intestinal ameba Entamoeba dispar, a feature used for clinical diagnosis. As a precursor to defining the roles amebic cell killing and phagocytic ability play in pathogenesis, this project’s goal is to delineate the molecular mechanisms underlying these processes. For this we are using flow cytometry and microscopy-based phagocytosis assays, confocal microscopy to localize epitope-tagged recombinant proteins, and a variety of biochemical and molecular techniques. Our studies have shown that E. histolytica kills host cells by inducing apoptosis, and ingests apoptotic cells more efficiently than healthy or necrotic cells. Furthermore, inhibition of the major amebic surface adhesin, which completely blocks adherence and cell killing, does not prevent ingestion of apoptotic cells, implicating at least one additional receptor in the recognition and clearance of killed cells. By purifying amebic phagosomes and using mass spectrometry, several candidate phagocytosis receptors have been identified and these proteins are currently being characterized. These studies promise to yield a greater understanding of how E. histolytica causes disease and may suggest improved methods for treatment and prevention of amebiasis.

Cryptosporidium species cause severe diarrhea in both immunocompetent and immunocompromised individuals. The infectious oocysts are resistant to standard water treatment methods and Cryptosporidium has been associated with numerous waterborne epidemics including one involving more than 400,000 Milwaukee residents in 1993. Nitazoxanide is the only available treatment for cryptosporidiosis, but it is ineffective for immunocompromised patients in whom infection can be fatal. Though new drugs are badly needed, drug development has been impeded by an inability to continuously culture the parasite in vitro. Within the host Cryptosporidium is dependent on anaerobic metabolism, making its fermentation enzymes attractive drug targets. Our preliminary studies indicate that a bifunctional acetaldehyde-alcohol dehydrogenase E (CpAdhE) catalyzes two terminal fermentation reactions. The AdhE enzymes are structurally distinct from human dehydrogenases, and contain separate acetaldehyde and alcohol NAD+-dependent dehydrogenase domains. Many gram-negative and gram-positive bacteria as well as several intestinal protozoa have AdhE enzymes, and an AdhE is essential for anaerobic growth of Eschericia coli. In this work, we are using a transgenic model to circumvent the need to cultivate Cryptosporidium and validate AdhE as a drug target for cryptosporidiosis. Genetic complementation of an E. coli AdhE mutant with recombinant CpAdhE and assays for restoration of anaerobic growth are being used to verify the function of CpAdhE. To assay NAD+-dependent aldehyde and alcohol dehydrogenase activities in bacterial lysates, a spectrophotometer is used to follow reduction of NAD+ to NADH. The long-term goal of this project is to use this spectrophotometric assay to conduct a small molecule screen for CpAdhE inhibitors. Cryptosporidium parvum’s dependence on CpAdhE for growth and development will be determined using an assay for C. parvum development in tissue culture. We expect these studies to yield important data on Cryptosporidium metabolism and to identify compounds that may be useful for treatment of this deadly parasitic infection.

Model of sequential cell killing and phagocytosis by E. histolytica

E. histolytica (green) ingests apoptotic (B) but not viable (A) Jurkat lymphocytes (red).