Monteiro Jomar Patrício, Clemons Karl V, Mirels Laurence F, Coller John A, Wu Thomas D, Shankar Jata, Lopes Catalina R, Stevens David A
Department of Medicine, Division of Infectious Diseases, Santa Clara Valley Medical Center, San Jose, CA, USA.
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA, USA.
Microbiology (Reading). 2009 Aug;155(Pt 8):2795-2808. doi: 10.1099/mic.0.027441-0. Epub 2009 Apr 30.
Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25 % of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.
巴西副球孢子菌是一种温度双态真菌，可引起拉丁美洲最常见的系统性真菌病。感染由宿主吸入分生孢子或菌丝片段引发，随后进一步分化为酵母形态。关于两种形态在培养中的多个生长时间点的基因表达信息很少被研究。在此，我们报告构建了一个基因组DNA微阵列，覆盖该生物体约25%的基因组，并利用其鉴定体外生长过程中的基因和基因表达模式。从随机剪切的基因组DNA（gDNA）和已知对照基因中克隆、扩增的插入片段被打印到载玻片上，以生成一个超过12000个元件的微阵列。为了检测基因表达，从在半限定培养基中培养5天、8天和14天的菌丝体或酵母培养物中提取并扩增mRNA。主成分分析和层次聚类表明，酵母基因表达谱与菌丝体的有很大差异，尤其是在早期时间点，并且随着时间的推移，菌丝体基因表达的变化小于酵母中的基因表达。在酵母中上调的基因被发现编码参与蛋氨酸/半胱氨酸代谢、呼吸和代谢过程（糖、氨基酸、蛋白质和脂质）、转运蛋白（小肽、糖、离子和毒素）、调节蛋白和转录因子的蛋白质。参与细胞分裂、蛋白质分解代谢、核苷酸生物合成以及毒素和糖转运等过程的菌丝体基因表现出差异表达。将测序的克隆与荚膜组织胞浆菌和波萨达斯球孢子菌的基因组序列进行比较，以评估跨物种潜在的共同途径，如硫和脂质代谢、氨基酸转运蛋白、转录因子以及可能与毒力相关的基因。我们还分析了培养过程中随时间变化的基因表达，发现虽然转座元件和呼吸途径的成分倾向于随时间增加表达，但编码核糖体结构蛋白和蛋白质分解代谢的基因倾向于随时间急剧降低表达，尤其是在酵母中。这些发现扩展了我们对巴西副球孢子菌在培养生长过程中不同形态形式的认识。
