Studies of the Pathogenesis of Amebiasis, Including Characterization of a Low Molecular Weight Protein Tyrosine Phosphatase

Entamoeba histolytica is a protozoal eukaryotic intestinal parasite of humans, and is endemic in developing countries or under circumstances where hygiene is poor. In the Petri lab we are interested in amebic virulence: the how and why of the ability of this parasite to cause disease. We chose RNAi to attempt to knock down gene expression in E. histolytica, and developed the short hairpin RNA interference system as a tool to reduce protein expression in order to study the function of virulence-involved genes. We used an episomal vector-based system utilizing the U6 promoter to drive expression of 29- basepair short hairpin RNAs (shRNAs) to knock down expression of four unrelated genes: PATMK, Igl, URE3-BP and EhC2A. PATMK levels were reduced by 99%, Igl levels by 72%, URE3-BP by 89%, and EhC2A by 97%. This shRNA system using the U6 promoter to drive expression of shRNAs is effective at knocking down gene expression in E. histolytica, and thus is a useful tool to study gene function in this organism, especially of virulence factors. We have partially characterized an E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The equivalent mammalian protein regulates cell proliferation, motility, and adhesion. These attributes are also needed for amebic virulence. The closest matches to this enzyme are found in plant or Bacteroides species, and are ~50 0dentical. Several different structures for this amebic tyrosine phosphatase have been solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it is expressed at low but detectable levels, and can be iii detected by immunoprecipation followed by immunoblotting. When overexpressed as an epitope-tagged protein from an amebic expression vector, the LMW-PTP can be detected by immunoblotting without an immunoprecipitation step, and LMW-PTP-overexpressing transfectant amebae were used to visualize the cellular location of this protein. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of possible substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag for overexpression in amebae.

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