Steele M I, Kuhls T L, Nida K, Meka C S, Halabi I M, Mosier D A, Elliott W, Crawford D L, Greenfield R A
Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City 73126, USA.
Infect Immun. 1995 Oct;63(10):3840-5. doi: 10.1128/iai.63.10.3840-3845.1995.
Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.
随着微小隐孢子虫的子孢子侵入肠道上皮细胞并随后发育为无性和有性生命阶段，微小隐孢子虫成功寄生需要宿主和原生动物细胞膜的多次破坏。为了鉴定可能在这些膜改变中起作用的隐孢子虫蛋白，溶血活性被用作筛选微小隐孢子虫基因组表达文库的标记。鉴定出一个稳定的溶血克隆（H4），其含有一个5.5 kb的隐孢子虫基因组片段。H4上编码的溶血活性被定位到一个1 kb的区域，该区域包含一个完整的690 bp开放阅读框（hemA），以一个常见的终止密码子结尾。在含有H4的大细胞中表达了一种21 kDa的质粒编码重组蛋白。仅包含hemA一部分的H4亚克隆在血琼脂上不诱导溶血，也不促进重组蛋白在大细胞中的表达。对从脱囊的子孢子和感染严重联合免疫缺陷成年小鼠的肠道中分离的总RNA进行逆转录酶介导的PCR分析表明，hemA在隐孢子虫生命周期中被积极转录。
