Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville, Vic. 3010, Australia.
Mol Microbiol. 2011 Apr;80(2):471-80. doi: 10.1111/j.1365-2958.2011.07584.x. Epub 2011 Mar 8.
Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca(2+) /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Δcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Δcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Δcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Δcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Δcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca(2+) influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.
利什曼原虫寄生虫在感染其哺乳动物宿主时必须适应高温和其他环境应激。这些环境线索是如何被感知的还知之甚少。在这项研究中，我们表明钙摄取对于寄生虫在 34-37°C 的耐热性是必需的。为了鉴定钙流入的潜在下游靶标，生成了一种缺失钙（Ca 2+ ）/钙调蛋白依赖性丝氨酸/苏氨酸特异性磷酸酶的必需调节亚基（CnB）的 L. major 主要突变体，钙调神经磷酸酶。Δcnb 突变体在 27°C 时与野生型寄生虫生长良好且正常分化为感染性的循环前鞭毛体。然而，当暴露于高温（34°C）时，Δcnb 寄生虫失去活力，并且对内质网和膜应激高度敏感，内质网和膜应激分别由衣霉素和固醇和鞘脂生物合成抑制剂诱导。Δcnb 前鞭毛体被巨噬细胞内化，但它们向适应热的无鞭毛体阶段的分化被延迟，并且由此产生的寄生虫无法增殖。引人注目的是，易感的 BALB/c 小鼠完全清除了Δcnb 寄生虫。用 CnB 互补Δcnb 寄生虫恢复了在巨噬细胞和动物模型中对环境应激的早期和长期适应性寄生虫反应的耐热性和感染力。我们的研究结果表明，钙流入和钙调神经磷酸酶信号传导对于寄生虫在哺乳动物宿主中遇到的早期和长期适应环境应激的反应都是必需的。
