UC San Diego

Amebiasis, defined as invasive intestinal or extra- intestinal infection with the protozoan parasite Entamoeba histolytica, is a major cause of morbidity in developing countries and the second leading cause of death from parasitic disease worldwide. More than 500 million people worldwide are infected with Entamoeba, causing about 50 million cases of invasive amebiasis and more than 50,000 deaths every year. Based upon biochemical, immunological, and genetic studies, E. histolytica, has been reclassified into two morphologically identical, but genetically distinct species: E. histolytica, which is potentially invasive and E. dispar, which is not. Cysteine proteinases are considered key virulence factors of the protozoan parasite, E. histolytica, and play a central role in tissue invasion and disruption of host defenses. Of all the reported virulence factors found in E. histolytica to date, only cysteine proteinases are encoded by unique genes, with at least two of the cysteine proteinase genes absent from E. dispar strains (EhCP1 and EhCP5). The main focus of this dissertation tested the hypothesis that, EhCP1, which is unique to E. histolytica, plays a crucial role in amebic invasion: Specific Aim 1: Tested the hypothesis that E. histolytica Cysteine Proteinase 1 (EhCP1) differs from the other cysteine proteinases in its specificity for substrates. In these studies I: a) Expressed active recombinant EhCP1 and purified it to homogeneity. Recombinant pro-EhCP1 was expressed in E. coli and an optimized refolding protocol yielded active enzyme. b) Characterized EhCP1 by its pH optimum, Km for synthetic substrates, and active site mapping. The purified recombinant enzyme had a Km of approximately 2 [mu]M with the fluorogenic peptidyl substrate Z-Arg-Arg- AMC. Its pH optimum was 6.0-6.5, but retained activity > 50% between pH 6.0 and 9.0. We mapped the specificity of the P1-P4 subsites of the active site cleft using a positional-scanning synthetic tetra-peptide combinatorial library. Arginine was strongly preferred at the P2 position, an unusual specificity among Clan CA proteinases. c) Determined the specificity of EhCP1 for synthetic and physiological (biological) substrates. Recombinant EhCP1 cleaved C3, human IgG and pro-IL-18 in a time- and dose-dependent manner similar to purified native proteinases. Aim 2: Tested the hypothesis that EhCP1 differs in its release or localization from other cysteine proteinases by: a) Identifying and quantifying the major released cysteine proteinases by quantitative ELISA. EhCP1 is one of the major released cysteine proteinases as shown with a sensitive ELISA using monoclonal or polyclonal antibody to recombinant forms of EhCP1, EhCP2, EhCP3, and EhCP5 to quantify released proteinases. EhCP2 was the major released cysteine proteinase followed by EhCP5>EhCP1 >EhCP3. b) Determining the intracellular localization of EhCP1 by fluorescence, confocal and electron microscopy. EhCP1 localized to large cytoplasmic vesicles, separate from those containing EhCP3. Aim 3: Tested the hypothesis that inhibition of EhCP1 blocked or significantly diminished invasion. In these studies I compared the effect of specific cysteine proteinase inhibitors on in vivo invasion in the human intestinal xenograft model in SCID mice. Both pathology and quantification of the number of invading ameba by a sensitive PCR assay demonstrated that the specific irreversible vinyl sulfone cysteine proteinase inhibitors, K11777 and WRR 483, almost completely blocked amebic invasion in the human intestinal xenograft model. Thus, EhCP1 is a major released cysteine proteinase of invasive E. histolytica, which is important in both invasion and disruption of the host response