Neutral cysteine proteinases of E. histolytica are the major extracellular enzymes which first interact with host defenses. Experimental evidence supporting the role of neutral cysteine proteinases in amebic invasion includes the ability of the purified enzyme from pathogenic strains (E. histolytica) to degrade components of the extracellular matrix. The proteinase also interacts directly with the immune system by degrading IgA and activating complement by specifically cleaving C3 and C5, and producing and degrading the anaphylatoxins, C3a and C5a. E. histolytica strains have a unique gene (acp 1) encoding the neutral cysteine proteinase, higher levels of proteinase-specific mRNA, increased proteinase release, and enhanced cytopathic effect. We propose to study the structure and function of cysteine proteinases with the following Specific Aims: 1.) To characterize the extracellular neutral cysteine proteinase biochemically by: a.) Comparison of the peptide sequences of the purified enzymes to the derived amino acid sequences of the cloned genes. b.) Evaluation of the pH optima, stability, and substrate specificity of the enzymes for synthetic and structural substrates. c.) Purification, characterization, and comparison of active recombinant enzymes. d.) Production of monoclonal antibodies that distinguish between the different enzymes (ACP 1, 2, and 3). 2.) To evaluate the processing and release of the extracellular cysteine proteinase which interacts with host defenses by: a.) Comparing the processing of the proenzymes of ACP 1, 2, and 3. b.) Assessing the requirement of an acid pH for activation of the cysteine proteinases. c.) Evaluating whether prosequences are required for production of active enzymes. d.) Comparing the intra- and extracellular levels of the three enzymes. e.) Measure the effect of exposure to cellular targets or extracellular matrix on the release of proteinases. 3.) To extend our initial observations on the interactions of the proteinase with the host immune system by: a.) Examining the ability of the extracellular cysteine proteinases to cleave kininogen, liberating the vasodilatory peptide, bradykinin, which could promote vascular invasion. b.) Analyzing the cleavage of IgG bound to the ameba membrane and in the fluid-phase. 4.) To directly evaluate the role of cysteine proteinases in invasion in vivo by: a.) Evaluating the effect of cysteine proteinase inhibitors on cecal invasion in a gerbil model of intestinal amebiasis. b.) Evaluating the effect on cysteine proteinase inhibitors on the establishment of liver abscesses in gerbils. These studies should further our understanding of an important virulence factor of E. histolytica at both the biochemical and genetic level. The identification of novel features of the amebic enzymes may reveal potential targets for immunotherapy or chemotherapy in the future.
