Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

中文翻译：

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GRASP 负向调节利什曼原虫毒力因子 gp63 的分泌


金属蛋白酶-gp63是利什曼原虫分泌的毒力因子。然而，利什曼原虫的分泌途径尚不明确。在这里，我们克隆并表达了利什曼原虫的 GRASP 同源物。我们发现利什曼原虫表达一种 58 kDa 蛋白 (LdGRASP) 的 GRASP 同源物，该蛋白定位于利什曼原虫的 LdRab1 和 LPG2 阳性高尔基体区室中。发现 LdGRASP 与 COPII 复合物、LdARF1、LdRab1 和 LdRab11 结合，表明其在利什曼原虫内质网和高尔基体运输中的作用。为了确定 LdGRASP 的功能，我们使用 CRISPR-Cas9 生成了 LdGRASP 敲除寄生虫。我们在 Ld:GRASPKO 寄生虫中发现高尔基体碎片。我们的结果表明，与 Ld:WT 细胞相比，非 GPI 锚定的 gp63 向细胞表面的转运增强，导致 Ld:GRASPKO 寄生虫中这种形式的 gp63 的分泌量更高。相反，我们发现 Ld:GRASPKO 寄生虫中 GPI 锚定的 gp63 向细胞表面的转运被阻断，从而抑制其分泌。 Ld:GRASPKO 寄生虫中 LdRab1 或 LdSar1 显性失活突变体的过度表达显着阻断了非 GPI 锚定的 gp63 的分泌。有趣的是，我们发现与 Ld:WT 和 Ld:GRASPKO 寄生虫相比，过表达 Ld:GRASP-GFP 的转基因寄生虫在巨噬细胞中的存活率显着降低。这些结果表明，LdGRASP 差异调节利什曼原虫中的 Ldgp63 分泌途径。