Role of CTR4 in the Virulence of Cryptococcus neoformans.
Role of CTR4 in the Virulence of Cryptococcus neoformans.
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DOI：
10.1128/mbio.00285-12
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发表时间：
2012
期刊：
影响因子：
6.4
通讯作者：
Williamson PR
中科院分区：
文献类型：
--
作者：
Waterman SR;Park YD;Raja M;Qiu J;Hammoud DA;O'Halloran TV;Williamson PR
虽然研究已经确定了铁等金属在微生物发病中的重要贡献，但铜等其他过渡金属的作用大多仍不清楚。最近的证据表明，在小鼠模型和人类患者队列中，基于CUF1铜调节因子的作用，新生隐球菌的毒力需要铜的动态平衡。金黄色葡萄球菌是一种重要的真菌病原体，每年估计导致60万人死于艾滋病。在目前的研究中，我们发现缺乏依赖CUF1的铜转运蛋白CTR4的新生隐球菌突变体在37℃的富含介质中正常生长，但在小鼠模型中降低了巨噬细胞的存活率和毒力。这种存活率和毒力的下降被追溯到营养限制条件下的生长缺陷。使用全长CTR4-荧光融合报告构建的表达研究表明，在巨噬细胞、脑和肺中有强劲的表达，后者通过体外荧光成像显示。用电感耦合质谱仪(ICPMS)检测了在特定条件下培养的真菌细胞和从脑组织中回收的真菌细胞的铜配额，表明在富铜条件下CTR4与细胞金属硫蛋白的联合保护铜的作用。综上所述，这些数据表明，CTR4在铜相关的动态平衡以及随后的真菌毒力中发挥了作用。新生隐球菌是一种重要的全球性真菌病原体，铜稳态是微生物发病机制中相对未被探索的一个方面，可能导致新的治疗方法。以前的研究表明，Ctr4铜转运蛋白的表达水平与实体器官移植患者队列中脑膜脑炎的发生有关，但Ctr4在哺乳动物发病机制中的直接作用尚未得到证实。本研究利用了一株CTR4ctr4突变株，该突变株揭示了Δ在小鼠感染新生梭菌中的重要作用，并将该基因产物与铜的动态平衡控制和营养限制条件下的生长联系起来。CTR4在真菌感染期间的强健表达水平被用来利用体外荧光成像来证明CTR4的表达与肺隐球菌病的关系。综上所述，这些研究首次直接证明了铜转运蛋白在真菌疾病中的作用，并为进一步研究隐球菌的基因表达和发病机制提供了体外成像工具。
While research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeostasis in the virulence of Cryptococcus neoformans based on a role for a CUF1 copper regulatory factor in mouse models and in a human patient cohort. C. neoformans is an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that a C. neoformans mutant lacking the CUF1-dependent copper transporter, CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies using a full-length CTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown by ex vivo fluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective function of CTR4 in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role for CTR4 in copper-related homeostasis and subsequently in fungal virulence. Crytococcus neoformans is a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a Δctr4 mutant strain which revealed an important role for CTR4 in C. neoformans infections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels of CTR4 during fungal infection were exploited to demonstrate expression and lung cryptococcal disease using ex vivo fluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide an ex vivo imaging tool for further study of cryptococcal gene expression and pathogenesis.