Department of Medical Microbiology and Immunology, University of Wisconsin—Madison, Madison, Wisconsin, USA.
Infect Immun. 2012 Jan;80(1):55-61. doi: 10.1128/IAI.05543-11. Epub 2011 Oct 17.
Toxoplasma gondii is an obligate intracellular parasite that uses immune cells to disseminate throughout its host. T. gondii can persist and even slowly replicate in activated host macrophages by reducing the antimicrobial effects of molecules such as nitric oxide (NO). A T. gondii patatin-like protein called TgPL1 was previously shown to be important for survival in activated macrophages. Here we show that a T. gondii mutant with a deletion of the TgPL1 gene (ΔTgPL1) is degraded in activated macrophages. This degradation phenotype is abolished by the removal of NO by the use of an inducible NO synthase (iNOS) inhibitor or iNOS-deficient macrophages. The exogenous addition of NO to macrophages results in reduced parasite growth but not the degradation of ΔTgPL1 parasites. These results suggest that NO is necessary but not sufficient for the degradation of ΔTgPL1 parasites in activated macrophages. While some patatin-like proteins have phospholipase A2 (PLA2) activity, recombinant TgPL1 purified from Escherichia coli does not have phospholipase activity. This result was not surprising, as TgPL1 contains a G-to-S change at the predicted catalytic serine residue. An epitope-tagged version of TgPL1 partially colocalized with a dense granule protein in the parasitophorous vacuole space. These results may suggest that TgPL1 moves to the parasitophorous vacuole to protect parasites from nitric oxide by an undetermined mechanism.
刚地弓形虫是一种专性细胞内寄生虫，它利用免疫细胞在宿主中传播。刚地弓形虫可以通过减少一氧化氮（NO）等分子的抗菌作用在激活的宿主巨噬细胞中持续存在甚至缓慢复制。先前已经表明，一种叫做 TgPL1 的刚地弓形虫类脂肪酶蛋白对于在激活的巨噬细胞中存活很重要。在这里，我们显示出 TgPL1 基因缺失的刚地弓形虫突变体（ΔTgPL1）在激活的巨噬细胞中被降解。通过使用诱导型一氧化氮合酶（iNOS）抑制剂或 iNOS 缺陷型巨噬细胞去除 NO，可以消除这种降解表型。将 NO 外加到巨噬细胞中会导致寄生虫生长减少，但不会导致 ΔTgPL1 寄生虫的降解。这些结果表明，NO 对于激活的巨噬细胞中 ΔTgPL1 寄生虫的降解是必要的，但不是充分的。虽然一些类脂肪酶蛋白具有磷脂酶 A2（PLA2）活性，但从大肠杆菌中纯化的重组 TgPL1 没有磷脂酶活性。这一结果并不奇怪，因为 TgPL1 在预测的催化丝氨酸残基处含有 G 到 S 的变化。TgPL1 的表位标记版本与寄生空泡空间中的致密颗粒蛋白部分共定位。这些结果可能表明，TgPL1 通过未知机制移动到寄生空泡中，以保护寄生虫免受一氧化氮的侵害。
