Laboratory of Leishmaniasis Prof. Dr. Ralph Lainson, Evandro Chagas Institute, Department of Health Surveillance, Ministry of Health, Ananindeua 67030-000, Pará, Brazil; Federal University of Para, Institute of Biological Sciences, Laboratory of Structural Biology, Belém 66095-110, Pará, Brazil.
Federal University of Para, Institute of Biological Sciences, Laboratory of Structural Biology, Belém 66095-110, Pará, Brazil.
Parasitol Int. 2021 Aug;83:102347. doi: 10.1016/j.parint.2021.102347. Epub 2021 Apr 19.
Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/- 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/- 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.
利什曼原虫是一种专性细胞内寄生虫，主要栖息在巨噬细胞内。寄生虫在宿主细胞内的破坏是感染控制的基本机制。此外，抑制巨噬细胞的杀利什曼原虫活性似乎与某些物种通过耗尽精氨酸来抑制一氧化氮（NO）产生的能力有关。一些利什曼原虫物种具有从组成型一氧化氮合酶样酶（cNOS 样）产生 NO 的能力。然而，以前尚未描述 cNOS 样在利什曼原虫中的定位。因此，本研究旨在定位利什曼原虫（利什曼原虫）和利什曼原虫（Viannia）巴西利什曼原虫的前鞭毛体中的 cNOS 样酶和 NO 产生。通过流式细胞术最初定量了 NO 产生，该结果表明 L.（L.）亚马逊（GMFC=92.17 +/- 4.6）和 L.（V.）巴西利什曼原虫（GMFC=18.89 +/- 2.29）之间的 NO 产生存在显著差异（P<0.05）。免疫印迹分析显示 cNOS 表达更多，L.（L.）亚马逊与 L.（V.）巴西利什曼原虫相比。随后，在靠近囊泡、鞭毛囊和线粒体的鞭毛体中观察到 cNOS 样免疫标记，并且似乎有小簇的颗粒与囊泡融合，提示糖体，过氧化物酶样细胞器，将糖酵解途径分隔在原生动物寄生虫中。此外，共聚焦显微镜分析显示 cNOS 样和 GAPDH 的共定位，GAPDH 是糖体的特异性标志物。因此，L.（L.）亚马逊产生的 NO 量大于 L.（V.）巴西利什曼原虫，并且两种物种都在糖体内部存在 cNOS 样酶。
