Host-Pathogen Interactions of Intestinal Protozoa
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.</br>
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. This project’s goal is to delineate the molecular mechanisms underlying amebic killing and phagocytosis of host cells. 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 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.</br>
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, and by lack of economic incentive to develop new molecules for treatment of this disease, which predomantly affects people in developing nations. In order to address the clinical need for better anti-cryptosporidial drugs while acknowledging the economic reality of who this disease affects, we are taking a drug re-purposing approach. That is, we have focused our efforts on identification of drugs that are already approved or for which a substantial economic investment has already been made for other diseases. To enable this approach, we have developed and optimized a high-throughput cell-based assay for in vitro growth of Cryptosporidium parvum in intestinal epithelial cells that utilizes automated liquid handling, microscopy and image analysis. We have used this assay to screen several small molecule libraries and identify novel inhibitors of C. parvum growth, several of which we are now developing as potential treatments for humans with cryptosporidiosis. Furthermore, since we have screened compounds with known bioactivities, there is the potential to learn a great deal about the biology of Cryptosporidium species based on what is already known about the newly identified growth inhibitors.
Sequential model of adherence, host cell killing, and phagocytosis by Entamoeba histolytica.
E. histolytica (green) ingests apoptotic (B) but not viable (A) Jurkat lymphocytes (red).
E. histolytica calreticulin (green) is present on the cell surface and functions as a phagocytosis receptor. Scale bar is 10 microns.
Cryptosporidium parvum infection of the human intestinal epithelial cell line HCT-8. C. parvum (green); DNA (blue); scale bar is 5 microns.
Heron BT, Sateriale A, Teixeira JE, Huston CD. Evidence for a novel Entamoeba histolytica lectin activity that recognizes carbohydrates present on ovalbumin. International Journal for Parasitology. 2011. 41:137-44. PMID: 20807536
Teixeira JE, Sateriale A, Bessoff KE, Huston CD. Control of Entamoeba histolytica adherence involves EhMSP-1, a M8 family surface metalloprotease with homology to leishmanolysin. Infection and Immunity. 2012. 80:2165-76. PMID: 22451519.
Vaithilingam A, Teixeira JE, Miller PJ, Heron BT, Huston CD. Entamoeba histolytica cell surface calreticulin binds human C1q and functions in amebic phagocytosis of host cells. Infection and Immunity. 2012. 80:2008-2018. PMID: 22473608.
Sateriale A, Vaithilingam A, Donnelly L, Miller P, Huston CD. Feed forward regulation of phagocytosis by Entamoeba histolytica. Infection and Immunity. 2012. 80:4456-4462. PMID: 23045476.
Bessoff K, Sateriale A, Lee KK, Huston CD. Drug repurposing screen reveals FDA-approved inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium parvum growth. Antimicrobial Agents and Chemotherapy. 2013. Feb 4 [ePub ahead of print]. PMID: 23380723.
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