Genetic Manipulation of Entamoeba Virulence

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DESCRIPTION (from the applicant's abstract): Cysteine proteinases are key virulence factors of E. histolytica and play a central role in tissue invasion and disruption of host defenses. We have shown that purified cysteine proteinases of E. histolytica degrade components of the extracellular matrix and cleave IgG, IgA, the C3 and C5 components of complement, and the anaphylatoxins, C3a and C5a, limiting the host response to amebic infection. E. histolytica and E. dispar are morphologically identical with highly homologous genomes including cysteine proteinase genes, but only E. histolytica can invade the host. We propose to test the hypothesis that surface and extracellular cysteine proteinases are critical for amebic invasion with the following Specific Aims: Aim 1: We will test the hypothesis that the cysteine proteinases, which are critical to invasion, differ in their location, release, or specificity for substrates. These studies will identify the major extracellular proteinases, express active, recombinant cysteine proteinases, and identify differences in specificity against peptide and physiological substrates. Aim 2: We will test the hypothesis that inhibition of the key cysteine proteinases will block invasion. These studies will compare the effect of specific peptide inhibitors, antisense constructs, and insertional proteinase mutants on invasion. Aim 3: We will test the hypothesis that complementation of cysteine proteinase expression in proteinase-deficient strains will restore virulence. We will use selectable expression vectors to over express specific cysteine proteinases in E. dispar and L6 to evaluate the effect on invasion. These studies should further our understanding of an important virulence factor of E. histolytica and establish the key cysteine proteinases, which are linked to invasion and could be targets of novel drug therapy in the future.