Fernández-Arenas Elena, Cabezón Virginia, Bermejo Clara, Arroyo Javier, Nombela César, Diez-Orejas Rosalía, Gil Concha
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.
Mol Cell Proteomics. 2007 Mar;6(3):460-78. doi: 10.1074/mcp.M600210-MCP200. Epub 2006 Dec 12.
The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.
白色念珠菌与巨噬细胞的相互作用被认为是全身性念珠菌病中产生充分免疫反应的关键步骤。我们建立了一种吞噬作用的体外模型，该模型包括一种鉴别内化和未内化酵母的差异染色程序。在优化获取摄入酵母富集群体的方案后，对这些细胞进行了全面的基因组学和蛋白质组学分析。蛋白质和mRNA均从同一样本中获取并并行分析。二维聚丙烯酰胺凝胶电泳与质谱联用揭示了巨噬细胞相互作用后共有132种差异表达的酵母蛋白种类。在这些种类中，鉴定出了67种独特的蛋白质。这是首次使用蛋白质组学方法研究白色念珠菌与巨噬细胞的相互作用。我们提供了证据表明，真菌通过改变属于不同途径的蛋白质表达，对吞噬体内的新环境做出快速的蛋白质反应。碳化合物代谢明显下调，加上脂质、脂肪酸、乙醛酸和三羧酸循环上调，表明酵母转变为饥饿模式。降解和解毒蛋白质机制有重要激活。互补的基因组学方法导致检测到与念珠菌毒力相关的特定途径。网络分析使我们能够生成巨噬细胞相互作用后念珠菌细胞死亡的假设模型，突出了肌动蛋白细胞骨架、线粒体和自噬在细胞凋亡调节中的相互联系。总之，基因组学、蛋白质组学和网络分析的结合是更好地理解复杂的宿主-病原体相互作用的有力策略。
