A CRYPTOSPORIDIUM-PARVUM GENOMIC REGION ENCODING HEMOLYTIC-ACTIVITY

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidi al sporozoites invade intestinal epithelial cells and subsequently dev elop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolyti c activity was used as a marker to screen a C. parvum genomic expressi on library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosp oridial genomic fragment was identified. The hemolytic activity encode d on H4 was mapped to a 1-kb region that contained a complete 690-bp o pen reading frame (hemA) ending in a common stop codon. A 21-kDa plasm id-encoded recombinant protein was expressed in maxicells containing H 4. Subclones of H4 which contained only a portion of hemA did not indu ce hemolysis on blood agar or promote expression of the recombinant pr otein in maxicells. Reverse transcriptase-mediated PCR analysis of tot al RNA isolated from excysted sporozoites and the intestines of infect ed adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.