University of Birmingham Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci
University of Birmingham Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci
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发表时间：
2010
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通讯作者：
Kerstin Voelz;S. Johnston;Julian C Rutherford;R. May
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作者：
Kerstin Voelz;S. Johnston;Julian C Rutherford;R. May
人类真菌病原体新型隐球菌和格特隐球菌会引起危及生命的中枢神经系统感染。隐球菌疾病的主要特征之一是病原体寄生在吞噬免疫效应细胞上的能力，这种现象与啮齿动物感染模型中的毒力密切相关。尽管吞噬细胞/隐球菌相互作用对疾病进展很重要，但目前测定巨噬细胞系统毒力的方法既耗时又低通量。在这里，我们介绍了两种广泛使用的隐球菌菌株的第一个稳定且完全表征的 GFP 表达衍生物：新型隐球菌血清型 A 型菌株 H99 和格特隐球菌血清型 B 型菌株 R265。两种菌株对环境和宿主应激条件的反应均未改变，并且在巨噬细胞模型系统中没有毒力缺陷。此外，我们报告了一种通过流式细胞术有效快速研究巨噬细胞寄生的方法的开发，该技术保留了当前方法的准确性，但速度提高了四倍。
The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/ Cryptococcus interactions to disease progression, current methods for assaying virulence in the macrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed.