van Mansfeld Rosa, de Been Mark, Paganelli Fernanda, Yang Lei, Bonten Marc, Willems Rob
Department of medical microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands.
Novo Nordisk Foundation center for Biosustainability, Technical University of Denmark, Copenhagen, Denmark.
PLoS One. 2016 Jun 23;11(6):e0158106. doi: 10.1371/journal.pone.0158106. eCollection 2016.
This study investigates adaptation of ST406, a prevalent P. aeruginosa clone, present in 15% of chronically infected cystic fibrosis (CF) patients in the Netherlands, in a newly infected CF patient during three years using whole genome sequencing (WGS), transcriptomics, and phenotypic assays, including biofilm formation. WGS-based phylogeny demonstrates that ST406 is genetically distinct from other reported CF related strains or epidemic clones. Comparative genomic analysis of the early (S1) and late (S2) isolate yielded 42 single nucleotide polymorphisms (SNPs) and 10 indels and a single 7 kb genomic fragment only found in S2. Most SNPs and differentially expressed genes encoded proteins involved in metabolism, secretion and signal transduction or transcription. SNPs were identified in regulator genes mexT and exsA and coincided with differential gene expression of mexE and mexF, encoding the MexE/F efflux pump, genes encoding the type six secretion system (T6SS) and type three secretion system (T3SS), which have also been previously implicated in adaptation of other P. aeruginosa strains during chronic infection of CF lungs. The observation that genetically different strains from different patients have accumulated similar genetic adaptations supports the concept of adaptive parallel evolution of P. aeruginosa in chronically infected CF patients. Phenotypically, there was loss of biofilm maturation coinciding with a significant lower level of transcription of both bfmR and bfmS during chronic colonization. These data suggest that the high-prevalent Dutch CF clone ST406 displays adaptation to the CF lung niche, which involves a limited number of mutations affecting regulators controlling biofilm formation and secretion and genes involved in metabolism. These genes could provide good targets for anti-pseudomonal therapy.
本研究利用全基因组测序（WGS）、转录组学和包括生物膜形成在内的表型分析方法，对荷兰15%的慢性感染囊性纤维化（CF）患者中普遍存在的铜绿假单胞菌克隆ST406，在一名新感染CF患者体内三年内的适应性进行了研究。基于WGS的系统发育分析表明，ST406在基因上与其他报道的CF相关菌株或流行克隆不同。对早期（S1）和晚期（S2）分离株的比较基因组分析产生了42个单核苷酸多态性（SNP）和10个插入缺失，以及一个仅在S2中发现的7 kb基因组片段。大多数SNP和差异表达基因编码参与代谢、分泌、信号转导或转录的蛋白质。在调节基因mexT和exsA中鉴定出SNP，这与编码MexE/F外排泵的mexE和mexF、编码VI型分泌系统（T6SS）和III型分泌系统（T3SS）的基因的差异表达一致，这些基因先前也与其他铜绿假单胞菌菌株在CF肺部慢性感染期间的适应性有关。来自不同患者的基因不同的菌株积累了相似的基因适应性这一观察结果支持了铜绿假单胞菌在慢性感染CF患者中适应性平行进化的概念。在表型上，在慢性定植期间，生物膜成熟丧失，同时bfmR和bfmS的转录水平显著降低。这些数据表明，高流行的荷兰CF克隆ST406表现出对CF肺生态位的适应性，这涉及影响控制生物膜形成和分泌的调节因子以及参与代谢的基因的有限数量的突变。这些基因可能为抗假单胞菌治疗提供良好的靶点。
