BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ENZYMES SECRETED BY LEISHMANIA

人体的基本细胞生物学、生物化学和分子生物学 病原体利什曼原虫作为寄生模型进行了研究。我们 重点集中在表征基本的生化功能和 几种独特的寄生虫分泌酶的基因结构， 以确定它们在寄生虫生存、生长和 传输 在这方面，L杜氏前鞭毛体（Pro）组成型 释放两种不同的分泌酸亚型（110和130 kDa） 磷酸酶（SAcP）的活性。我们分离并鉴定了 （SAcP-1和SAcP-2），其编码这些同工酶中的每一种。 其在 体外转录/翻译产物特异性地 用针对纯化的天然SAcP的抗体免疫沉淀。 SAcP-1和SAcP-2是在两个Ld中转录的单拷贝基因， 亲和无鞭毛体。这两个串联基因的位点是一个 单个约1.25 Mb大小的Ld染色体，该染色体也存在于其它染色体中 利什曼原虫表明其结构保守。此外，内脏 利什曼病患者显示具有针对 Ld SAcPs表明其在人类疾病中的存在。 在其他研究中， 分离并鉴定了Ld几丁质酶基因（CHI）。 成绩单 从Ld Pros中鉴定出100%的CHI，并且抗CHI肽血清反应于 用单个约52 kDa Ld Pro蛋白进行蛋白质印迹。CHI的轨迹 在Ld和其它染色体上定位到一条约450 kb的染色体上， 利什曼原虫表明其跨物种的结构保护 边界与贝茨博士合作，使用Ld CHI探针 来鉴定墨西哥利什曼原虫的同源基因， 从而导致人类皮肤疾病。最后，我们发现了一种新的 Ld Pros中约38 kDa的分泌核酸酶。基于其活性和 组成性分泌，我们认为它必须发挥重要作用， 寄生虫通过水解宿主衍生的 核酸

The basic cell biology, biochemistry and molecular biology of the human pathogen, Leishmania, are investigated as a model of parasitism. Our focus centers on characterizing both the basic biochemical functions and gene structure of several unique parasite secretory enzymes with an aim toward defining their essential roles in parasite survival, growth and transmission. In that regard, L. donovani (Ld) promastigotes (Pro) constitutively release two different isoforms (110 and 130 kDa) of secretory acid phosphatase (SAcP) in vitro. We isolated and characterized the genes (SAcP-1 and SAcP-2) which encode each of these isoenzymes. Their in vitro-transcription/translation products were specifically immunoprecipitated with antibodies against the purified, native SAcPs. SAcP-1 and SAcP-2 are single copy genes which are transcribed in both Ld Pro and amastigotes. The locus of these two tandemly linked genes is a single about 1.25 Mb-sized Ld chromosome which is also present in other Leishmania indicating its structural conservation. Moreover, visceral leishmaniasis patients were shown to possess specific antibodies against the Ld SAcPs indicating its presence in human disease. In other studies, an Ld chitinase gene (CHI) was isolated and characterized. Transcripts of CHI were identified from Ld Pros and anti-CHI peptide sera reacted on Western blots with a single about 52 kDa Ld Pro protein. The locus of CHI was mapped to a single about 450kb sized chromosome in Ld and other leishmanias indicating its structural conservation across species boundaries. In collaboration with Dr. Bates, the Ld CHI probe was used to identify the homologous gene in Leishmania mexicana, an organism which produces human cutaneous disease. Finally, we identified a new about 38 kDa secreted nuclease in Ld Pros. Based on its activity and constitutive secretion, we assume that it must play an essential role in parasite acquisition of nucleo-bases via hydrolysis of host-derived nucleic acids.