Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. Since this pathogen is categorized as a Class B bioterrorism agent, there is elevated priority to understand the molecular and cellular mechanisms that regulate its virulence. Two cellular processes that may play a role in pathogenicity are phagocytosis and PI 3-kinase-based signal transduction. Therefore, a better understanding of these processes may reveal new targets for anti-amoebic strategies. We propose a forward genetics approach that uses overexpression of proteins to identify genes that regulate phagocytosis and PI 3-kinase signaling.
In Aim 1, we will generate an E. histolytica cDNA library in an inducible E. histolytica expression system and transfect this library into E. histolytica trophozoites. We will then select for phagocytosis mutants and pinocytosis mutants from the population of transformants using screens based on the internalization of human red blood cells (loaded with a metabolic toxin, tubercidin) or on the internalization of a second metabolic toxin, methylenediphosphonate. Re-isolation of the expression plasmids from survivors and sequencing of the cDNA inserts will allow for the identification of genes that regulate these endocytic processes.
In Aim 2, we will select for wortmannin-resistant mutants from the population of transformants. Wortmannin is an inhibitor of PI 3-kinases and cells that express increased levels of wortmannin targets or their downstream effectors should tolerate higher levels of the drug. Identification of cDNAs overexpressed in survivors will reveal targets of wortmannin and downstream effectors of PI 3-kinases. This will provide a global view of PI 3-kinase signaling networks in E. histolytica. Completion of these studies will provide significant insight into the genes that regulate phagocytosis and PI 3-kinase signaling, important virulence functions in E. histolytica. Completion of these studies will also result in the development of a new research tool, an inducible cDNA expression library.

Public Health Relevance

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. Since this pathogen is also categorized as a Class B bioterrorism agent, there is elevated priority to understand the molecular and cellular mechanisms that regulate its virulence. The proposed studies will identify genes that regulate phagocytosis and signal transduction in this organism. Since these cellular processes are important to E. histolytica virulence, this work will significantly enhance our understanding of pathogenicity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI081100-01
Application #
7573223
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Joy, Deirdre A
Project Start
2009-06-19
Project End
2011-05-31
Budget Start
2009-06-19
Budget End
2010-05-31
Support Year
1
Fiscal Year
2009
Total Cost
$212,483
Indirect Cost
Name
Clemson University
Department
Biology
Type
Schools of Earth Sciences/Natur
DUNS #
042629816
City
Clemson
State
SC
Country
United States
Zip Code
29634