Nunes Luiz R, Costa de Oliveira Regina, Leite Daniela Batista, da Silva Vivian Schmidt, dos Reis Marques Everaldo, da Silva Ferreira Márcia Eliana, Ribeiro Diógenes Custódio Duarte, de Souza Bernardes Luciano Angelo, Goldman Maria Helena S, Puccia Rosana, Travassos Luiz R, Batista Wagner L, Nóbrega Marina Pasetto, Nobrega Francisco G, Yang Ding-Yah, de Bragança Pereira Carlos A, Goldman Gustavo H
Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Brazil.
Eukaryot Cell. 2005 Dec;4(12):2115-28. doi: 10.1128/EC.4.12.2115-2128.2005.
Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.
巴西副球孢子菌是一种与副球孢子菌病（PCM）相关的双相真菌，PCM是一种在南美洲流行的系统性真菌病。在人类中，感染始于吸入真菌繁殖体，这些繁殖体到达肺上皮并转化为酵母寄生形式。因此，菌丝体到酵母的转变特别令人感兴趣，因为转化为酵母对于感染至关重要。我们使用了一种携带该真菌4692个基因序列的巴西副球孢子菌生物芯片，来监测菌丝体到酵母形态转变的几个时间点（从5小时到120小时）的基因表达。结果显示，共有2583个基因在至少一个实验时间点表现出统计学上的显著调节。在鉴定出的基因同源物中，一些编码参与氨基酸分解代谢、信号转导、蛋白质合成、细胞壁代谢、基因组结构、氧化应激反应、生长控制和发育的酶。通过实时逆转录PCR独立验证了20个基因的表达模式，揭示了两种方法获得的数据之间具有高度相关性。一个编码4-羟基苯丙酮酸双加氧酶（4-HPPD）的基因在菌丝体到酵母的分化过程中高度过表达，使用4-HPPD活性的特异性抑制剂NTBC [2-(2-硝基-4-三氟甲基苯甲酰基)-环己烷-1,3-二酮] 以及NTBC衍生物，能够在体外抑制该真菌致病酵母阶段的生长和分化。这些数据为进一步研究涉及NTBC及其衍生物作为抗PCM的新型化疗药物奠定了基础，并证实了基于阵列的方法在识别针对病原微生物开发替代治疗新靶点方面的潜力。
