Santic Marina, Akimana Christine, Asare Rexford, Kouokam Joseph C, Atay Safinur, Kwaik Yousef Abu
Department of Microbiology and Immunology, University of Louisville, College of Medicine, Louisville, KY 40202, USA.
Environ Microbiol. 2009 Jun;11(6):1473-81. doi: 10.1111/j.1462-2920.2009.01875.x. Epub 2009 Feb 10.
Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster-derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica-derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.
由于土拉弗朗西斯菌通过蜱、蚊子、马蝇和鹿蝇等节肢动物媒介传播到哺乳动物宿主中，最近的研究已将黑腹果蝇确立为一种节肢动物媒介模型系统。关于土拉弗朗西斯菌在节肢动物来源细胞内的细胞内命运，以及这种宿主 - 寄生虫适应性在该病原体感染哺乳动物进化过程中的作用，目前尚不清楚。在本报告中，我们探讨了土拉弗朗西斯菌新凶手亚种在黑腹果蝇来源的S2细胞中的细胞内运输。首先，我们表明，与土拉弗朗西斯菌全北区亚种来源的LVS菌株相似，土拉弗朗西斯菌新凶手亚种具有高度传染性，在S2细胞和成年果蝇体内呈指数级复制，并且以剂量依赖的方式对成年果蝇致命，而iglC、iglD和mglA突变体则存在缺陷。使用基于电子显微镜和荧光显微镜的吞噬体完整性分析，我们发现野生型菌株在感染后30 - 60分钟内逃入S2细胞的细胞质中，到6小时时，90%位于细胞质中。相比之下，iglC和iglD突变体约40 - 50%在6小时时逃入细胞质，而另一亚群则被包裹在多层囊泡（MLV）中。用自噬抑制剂甲基腺嘌呤预处理S2细胞可阻止MLV的形成，并且iglC和iglD突变细菌的所有液泡亚群都被包裹在单膜包围的液泡中。对土拉弗朗西斯菌在S2细胞内的内吞运输研究表明，细菌吞噬体与黑腹果蝇LAMP2 - GFP融合蛋白有短暂共定位，但与预先加载荧光葡聚糖的溶酶体没有共定位。我们的数据表明，携带iglC突变体的MLV获得了Lamp2和葡聚糖，而携带iglD突变体的MLV排除了这些晚期内体和溶酶体标记物。我们的数据表明，致病岛编码蛋白在调节人类巨噬细胞和节肢动物媒介来源细胞内运输中的作用存在关键差异。
