Molecular Mechanisms to Attenuate Leishmania Parasite

摘要：利什曼原虫引起人类疾病，其临床症状从自我愈合的皮肤损害到致命的内脏感染。此外，在流行地区，感染艾滋病毒的人特别容易感染利什曼原虫，一旦感染艾滋病毒，潜伏感染可能会重新激活。对这种寄生虫的细胞生物学和致病机制缺乏了解，使得控制这一严重的世界性健康风险的任务变得困难。在国内，这尤其令美国军事人员、他们的家人和其他前往或居住在疫情地区的旅行者感到担忧。为了找到控制这种病原菌的新方法，我们开始了了解寄生虫从无毒(前鞭毛体)向强(无鞭毛体)分化的机制的研究。利什曼原虫的膜蛋白或分泌蛋白与其抵抗宿主杀伤的防御机制有关。人们对利什曼原虫等锥虫寄生虫的分泌途径知之甚少，尤其是对糖基化/折叠以及膜和分泌蛋白的细胞内运输具有特别重要的作用。已知内质网驻留的伴侣蛋白在这些过程中发挥重要作用。我们认为，通过调节伴侣蛋白的表达来改变分泌过程可能会导致利什曼原虫毒力的减弱。我们已经克隆了杜氏利什曼原虫钙网蛋白和蛋白二硫键异构酶(PDI)等伴侣蛋白的几个同源物。过表达利什曼原虫钙网蛋白的P区可显著减少寄生虫分泌性酸性磷酸酶的分泌，这是一种可能的毒力因子，这一效应与寄生虫内活性酶的积累有关。这种寄生虫在人类巨噬细胞内的存活率降低。另一种必需的内质网伴侣PdI的活性部位和表达的突变也影响了S酸性磷酸酶的分泌。PdI对维持内质网内还原的环境和形成二硫键起重要作用。这些研究表明，伴侣蛋白表达的改变可以调节利什曼原虫假定的毒力因子的分泌，从而导致其传染性的减弱。

Summary: Leishmania parasite causes human disease with clinical symptoms ranging from-self healing cutaneous lesions to a fatal visceral infection. Additionally, in endemic areas, people infected with HIV are especially prone to Leishmania infection and latent infections can reactivate upon acquisition of HIV. The lack of understanding of cell biology and pathogenic mechanisms of this parasite makes the task of controlling this grave, worldwide health risk difficult. Closer to home, it is particularly of concern to U.S. military personnel, their families and other travellers visiting or living in the endemic areas. To find novel methods for control of this pathogen, we have initiated study to understand the mechanism of parasite differentiation from the avirulent (promastigote) to virulent (amastigote) form. Membrane or secretory proteins of Leishmania have been implicated for its defense mechanism against killing by the host. Very little is known about the secretory pathway of trypanosomatids parasites such as Leishmania in general with particular importance to gylcosyation/folding and intracellular transport of membrane and secretory proteins. The ER resident chaperone proteins are known to play an essential role in these processes. We argued that alteration of the secretion process via modulating the expression of chaperone proteins might result in attenuation of virulence in Leishmania. We have cloned several homologues of such chaperone proteins such as calreticulin and protein disulfide isomerase (PDI) from Leishmania donovani. Overexpression of the P-domain of Leishmania calreticulin resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases, a putative virulent factor.This effect is associated with an intracellular accumulation of active enzyme inside the parasite. Such parasites where shown to have decreased survival inside human macrophages. Mutations in the active site and expression of another essential ER chaperone PDI, which is important for maintaining reduced environment inside the ER and for the formation of disulphide bonds in addition to chaperone activity also affected the secretion of s-acid phosphatase (sAcP). These studies suggest that alteration in the chaperone protein expression can modulate the secretion of Leishmanial putative virulent factors, which can result in the attenuation of its infectivity.