Mordue Dana G, Scott-Weathers Casey F, Tobin Crystal M, Knoll Laura J
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706, USA.
Mol Microbiol. 2007 Jan;63(2):482-96. doi: 10.1111/j.1365-2958.2006.05538.x. Epub 2006 Dec 11.
The apicomplexan parasite Toxoplasma gondii is able to suppress nitric oxide production in activated macrophages. A screen of over 6000 T. gondii insertional mutants identified two clones, which were consistently unable to suppress nitric oxide production from activated macrophages. One strain, called 89B7, grew at the same rate as wild-type parasites in naïve macrophages, but unlike wild type, the mutant was degraded in activated macrophages. This degradation was marked by a reduction in the number of parasites within vacuoles over time, the loss of GRA4 and SAG1 protein staining by immunofluorescence assay, and the vesiculation and breakdown of the internal parasite ultrastructure by electron microscopy. The mutagenesis plasmid in the 89B7 clone disrupts the promoter of a 3.4 kb mRNA that encodes a predicted 68 kDa protein with a cleavable signal peptide and a patatin-like phospholipase domain. Genetic complementation with the genomic locus of this patatin-like protein restores the parasites ability to suppress nitric oxide and replicate in activated macrophages. A haemagglutinin-tagged version of this patatin-like protein shows punctate localization into atypical T. gondii structures within the parasite. This is the first study that defines a specific gene product that is needed for parasite survival in activated but not naïve macrophages.
顶复门寄生虫刚地弓形虫能够抑制活化巨噬细胞中一氧化氮的产生。对6000多个弓形虫插入突变体进行筛选，鉴定出两个克隆，它们始终无法抑制活化巨噬细胞产生一氧化氮。其中一个菌株称为89B7，在未活化的巨噬细胞中生长速度与野生型寄生虫相同，但与野生型不同的是，该突变体在活化巨噬细胞中会被降解。这种降解表现为随着时间推移，液泡内寄生虫数量减少，免疫荧光检测显示GRA4和SAG1蛋白染色消失，电子显微镜观察显示寄生虫内部超微结构出现囊泡化和破裂。89B7克隆中的诱变质粒破坏了一个3.4 kb mRNA的启动子，该mRNA编码一个预测的68 kDa蛋白，带有可裂解的信号肽和一个类patatin磷脂酶结构域。用这种类patatin蛋白的基因组位点进行基因互补，可恢复寄生虫抑制一氧化氮的能力，并在活化巨噬细胞中复制。这种类patatin蛋白的血凝素标记版本在寄生虫内显示出点状定位到非典型的弓形虫结构中。这是第一项定义了在活化但非未活化巨噬细胞中寄生虫存活所需的特定基因产物的研究。
