Transcriptomic comparison of wild-type and ptvR deletion mutant of Streptococcus pneumoniae D39. Streptococcus pneumoniae

Reversible or phenotypic tolerance to antibiotics within microbial populations has been implicated in treatment failure of chronic infections and development of persister cells. However, the molecular mechanisms regulating phenotypic drug tolerance are largely unknown. In this study, we identified a four-gene operon in Streptococcus pneumoniae that contributes to phenotypic tolerance to vancomycin (ptv). RNA-Seq, qRT-PCR, and luciferase reporter experiments revealed that transcription of the ptv operon (consisting of ptvR, ptvA, ptvB and ptvC) is induced by pneumococcal exposure to vancomycin. Further investigation showed that the transcription of the ptv operon is repressed by PtvR, a PadR-family repressor; transcriptional induction of the ptv operon by vancomycin is achieved by transcriptional de-repression of this locus. Importantly, de-repression of ptvABC significantly enhanced the levels of vancomycin-tolerant pneumococci. Gene fusion and deletion analyses revealed that PtvA, PtvB and PtvC are membrane-associated proteins, and all required for the PtvR-regulated phenotypic tolerance to vancomycin. Finally, gel-shifting test with recombinant PtvR uncovered that PtvR represses the transcription of the ptv operon by binding to two palindromic sequences of the ptv promoter. Together, the ptv locus represents a novel inducible system for pneumococcal response to stressful conditions, including those caused by antibiotics. Overall design: Pairwise comparison of wild-type and ptvR knockout mutant of Streptococcus pneumoniae D39 (RNA-seq), analyzing the effect of ptvR deletion on the transcriptome. It was performed with deep sequencing, using an Illumina HiSeq 2500 machine with 125 nt single-end reads.

微生物种群中存在的抗生素可逆性表型耐受性（phenotypic tolerance），与慢性感染治疗失败及持留菌（persister cells）的形成密切相关。然而，调控表型药物耐受性的分子机制目前仍不明晰。
本研究在肺炎链球菌（Streptococcus pneumoniae）中鉴定出一个与万古霉素（vancomycin）表型耐受性相关的四基因操纵子（operon），将其命名为ptv。通过RNA测序（RNA-Seq）、实时定量反转录聚合酶链式反应（qRT-PCR）以及荧光素酶报告基因实验，研究发现当肺炎链球菌暴露于万古霉素环境时，ptv操纵子（由ptvR、ptvA、ptvB和ptvC四个基因组成）的转录会被诱导激活。进一步研究表明，ptv操纵子的转录受到PadR家族阻遏蛋白（PadR-family repressor）PtvR的负调控；而万古霉素对ptv操纵子的转录诱导，正是通过解除该基因座（locus）的转录抑制实现的。值得注意的是，解除ptvABC的转录抑制可显著提升万古霉素耐受性肺炎链球菌的种群比例。基因融合与缺失实验分析显示，PtvA、PtvB和PtvC均为膜相关蛋白，且三者均为PtvR调控的万古霉素表型耐受性所必需。最后，通过重组PtvR蛋白开展的凝胶迁移实验（gel-shifting test）证实，PtvR可通过结合ptv启动子（promoter）的两段回文序列（palindromic sequences），抑制ptv操纵子的转录。综上，ptv基因座代表了一套全新的可诱导调控系统，用于肺炎链球菌应对包括抗生素胁迫在内的多种应激环境。
实验整体设计：对肺炎链球菌D39的野生型（wild-type）与ptvR敲除突变株（knockout mutant）进行两两比对分析（RNA-seq），以探究ptvR基因缺失对细菌转录组（transcriptome）的影响。本次测序采用Illumina HiSeq 2500测序平台，以125碱基单端读长（125 nt single-end reads）进行深度测序。