GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

IF 2.6 2区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Molecular Microbiology Pub Date : 2024-05-01 Epub Date: 2024-04-01 DOI:10.1111/mmi.15255

Kamal Kumar, Rituparna Basak, Aakansha Rai, Amitabha Mukhopadhyay

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Abstract

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.

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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一种毒力因子。然而，利什曼原虫的分泌途径尚未明确。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 的 GRASP 同源物（LdGRASP），该蛋白定位于利什曼原虫中 LdRab1 和 LPG2 阳性的高尔基区。研究发现，LdGRASP 与 COPII 复合物、LdARF1、LdRab1 和 LdRab11 结合，表明它在利什曼病菌的 ER 和高尔基体转运中发挥作用。为了确定 LdGRASP 的功能，我们使用 CRISPR-Cas9 生成了 LdGRASP 基因敲除寄生虫。我们发现 Ld:GRASPKO 寄生虫的高尔基体发生了分裂。我们的结果表明，与 Ld:WT 细胞相比，Ld:GRASPKO 寄生虫体内非 GPI 锚定的 gp63 向细胞表面的转运增强，导致这种形式的 gp63 分泌增加。相反，我们发现在 Ld:GRASPKO 寄生虫中，GPI-锚定的 gp63 向细胞表面的运输受阻，从而抑制了其分泌。在 Ld:GRASPKO 寄生虫中过表达 LdRab1 或 LdSar1 的显性阴性突变体可显著阻止非 GPI-anchored 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明显下降。这些结果表明，LdGRASP 对利什曼原虫的 Ldgp63 分泌途径具有不同的调节作用。

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来源期刊

Molecular Microbiology 生物-生化与分子生物学

CiteScore

7.20

自引率

5.60%

发文量

132

审稿时长

1.7 months

期刊介绍： Molecular Microbiology, the leading primary journal in the microbial sciences, publishes molecular studies of Bacteria, Archaea, eukaryotic microorganisms, and their viruses. Research papers should lead to a deeper understanding of the molecular principles underlying basic physiological processes or mechanisms. Appropriate topics include gene expression and regulation, pathogenicity and virulence, physiology and metabolism, synthesis of macromolecules (proteins, nucleic acids, lipids, polysaccharides, etc), cell biology and subcellular organization, membrane biogenesis and function, traffic and transport, cell-cell communication and signalling pathways, evolution and gene transfer. Articles focused on host responses (cellular or immunological) to pathogens or on microbial ecology should be directed to our sister journals Cellular Microbiology and Environmental Microbiology, respectively.