Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK.
Cell Microbiol. 2012 Aug;14(8):1271-86. doi: 10.1111/j.1462-5822.2012.01798.x. Epub 2012 May 8.
Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.
利什曼原虫 ISP 是一种类似 ecotin 的天然肽类胰蛋白酶家族丝氨酸肽酶抑制剂，而这种酶在利什曼原虫基因组中不存在。这导致人们提出 ISP 抑制宿主丝氨酸肽酶的假说，我们最近的研究表明 ISP2 抑制中性粒细胞弹性蛋白酶，从而增强寄生虫在鼠巨噬细胞中的存活。在这项研究中，我们发现 ISP1 的丝氨酸肽酶抑制活性低于 ISP2，在前鞭毛体中，两者通常位于细胞质和鞭毛中。然而，在无鞭毛前鞭毛体中，ISP1 和 ISP2 在前半桥粒中大量积累，而 ISP2 则位于细胞膜表面。我们生成了一个缺乏三种 ISP 基因（Δisp1/2/3）的 L. major 突变体，并与 Δisp2/3 突变体进行了比较，以阐明 ISP1 的生理作用。在体外培养中，与 Δisp2/3 群体相比，Δisp1/2/3 突变体中含有更多的长鞭毛无鞭毛体、 nectomonad 和 leptomonad 前鞭毛体，运动能力降低，此外，它的特征是从鞭毛囊中释放出高水平的类外泌体囊泡。这些数据表明，ISP1 在鞭毛动态平衡中起主要作用，其破坏会影响分化和鞭毛囊中囊泡的动态。
