Windmüller Nadine, Witten Anika, Block Desirée, Bunk Boyke, Spröer Cathrin, Kahl Barbara C, Mellmann Alexander
Institute of Hygiene, University Hospital Muenster, Muenster, Germany.
Leibniz Institute for Arteriosclerosis, Muenster, Germany.
Int J Med Microbiol. 2015 Jan;305(1):38-46. doi: 10.1016/j.ijmm.2014.10.005. Epub 2014 Oct 29.
The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last ("late") isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (≥8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators.
囊性纤维化（CF）患者的肺部常常被金黄色葡萄球菌定植和/或感染数年，主要是被单一优势克隆株感染。为了在这种环境中长期存活，金黄色葡萄球菌在与宿主因子、抗生素及其他病原体相互作用的过程中需要进行适应。在此，我们使用RNA测序（RNA-Seq）和全基因组测序（WGS）研究了来自一名患者的同基因对金黄色葡萄球菌分离株的长期转录及基因组适应性。模拟体内条件，我们在收获RNA用于后续分析之前，使用人工痰液培养基培养金黄色葡萄球菌分离株。我们通过定量实时（qRT）-PCR确认了RNA-Seq数据，并额外研究了来自同一患者的中间分离株，这些分离株在CF气道中总共持续存在了13.2年。对第一个和最后一个（“晚期”）分离株进行的比较RNA-Seq分析显示，在持续存在13.2年后，晚期分离株存在显著差异。在有氧培养的两个分离株中表达的2545个基因中，有256个基因上调，161个基因下调，最小变化倍数为2倍（2f）。聚焦于25个高度（≥8f）上调（n = 9）或下调（n = 16）的基因，我们鉴定出了几个编码参与免疫逃避、细菌传播或分泌的毒力因子的基因（例如spa、sak和esxA）。此外，这些基因在需氧、微需氧和厌氧条件下表现出相似的表达趋势。对中间金黄色葡萄球菌分离株中高度上调或下调基因进行的进一步qRT-PCR实验显示，多年来基因表达模式不同。对晚期金黄色葡萄球菌分离株中差异表达基因及其上游区域进行测序分析，结果显示只有少数基因组改变。比较转录组分析揭示了适应性变化，主要影响参与宿主-病原体相互作用的基因。尽管潜在机制尚不清楚，但我们的结果表明，适应性过程是由局部因素而非全局调节因子的激活引发的，超出了基因组突变的范畴。
