A Cystic Fibrosis Pseudomonas aeruginosa Isolate Specialised to Catabolise Nucleic Acids

Purpose: Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). We provide an insight to the DNA auxotrophy of P. aeruginosa PASS4 isolate. Better understanding of P. aeruginosa adaptations in the CF lung environment can have a great impact in the development of specialised treatment regimes aimed at the eradications of P. aeruginosa infections. Methods: P. aeruginosa strains PAO1 and PASS4 were grown in minimal medium with either L-Asparagine or DNA as a carbon source, in biological triplicates. RNA was extracted and sequenced on Illumina HiSeq 1000 platform. The sequence reads that passed quality filters were analyzed using EdgePro and DESeq packages, as well as the Rockhopper tool. Results: We mapped > 10 million paired sequence reads per sample to the genome of P. aeruginosa PAO1 and identified a total of 576 genes differentially expressed by PASS4 when grown in DNA (P value < 0.01, log2 fold-change 1< to < -1), with 322 genes upregulated and 254 genes downregulated. There were a total of 423 genes differentially expressed by PAO1 when grown in DNA (P value < 0.01, log2 fold-change 1< to <-1), with 359 genes upregulated and 64 genes downregulated . A total of 129 transcripts displayed similar expression patterns in both organisms, with 112 being upregulated and 17 down-regulated. Conclusions: Our study identified that P. aeruginosa PASS4 was a purine auxotroph. Purine auxotropy may represent a viable microbial strategy for adaptation to DNA rich environments such as the CF lung. mRNA profiles of P. aeruginosa strains PAO1 and PASS4 grown in minimal media with either DNA or L-Asparagine as a sole carbon source were generated by paired end sequencing, in triplicate, using Illumina HiSeq.

目的：铜绿假单胞菌（Pseudomonas aeruginosa）是囊性纤维化（CF）患者发病与死亡的主要诱因。本研究解析了铜绿假单胞菌分离株PASS4的DNA营养缺陷型特性。深入理解铜绿假单胞菌在CF肺环境中的适应机制，可为开发靶向根除铜绿假单胞菌感染的专业化治疗方案提供重要支撑。
方法：将铜绿假单胞菌菌株PAO1与PASS4接种于以L-天冬酰胺或DNA为唯一碳源的基础培养基中，设置3次生物学重复。提取总RNA并在Illumina HiSeq 1000测序平台上进行测序。对通过质量过滤的测序读段，采用EdgePro、DESeq分析包及Rockhopper工具进行数据分析。
结果：每个样本的双端测序读段均比对至铜绿假单胞菌PAO1基因组，共鉴定出576个在以DNA为碳源培养下的PASS4菌株差异表达基因（P值<0.01，log₂折叠变化>1或<-1），其中322个基因上调表达，254个基因下调表达。在以DNA为碳源培养的PAO1菌株中，共鉴定出423个差异表达基因（P值<0.01，log₂折叠变化>1或<-1），其中359个基因上调表达，64个基因下调表达。两个菌株中共存在129个转录本呈现相似的表达模式，其中112个上调、17个下调。
结论：本研究证实铜绿假单胞菌PASS4为嘌呤营养缺陷型（purine auxotroph）。嘌呤营养缺陷型或许是微生物适应如CF肺这类富DNA环境的可行策略。本研究通过Illumina HiSeq平台完成双端测序，设置3次生物学重复，获取了以DNA或L-天冬酰胺为唯一碳源的基础培养基中培养的铜绿假单胞菌PAO1与PASS4菌株的mRNA表达谱。