Mechanisms of resistance to tetracyline in Streptococcus pneumoniae R6 mutants

Background This study aimed at characterizing determinants of resistance to tetracycline intrinsic to the genome of Streptococcus pneumoniae. Results Whole-genome sequencing of two S. pneumoniae R6 mutants resistant to tetracycline and cross-resistant to the fluoroquinolone ciprofloxacin revealed mutations in the gene rpsJ coding for the ribosomal protein S10 and in the operon coding for the ABC proteins PatA and PatB. Resistance reconstruction in a S. pneumoniae R6 tetracycline sensitive background confirmed the role of the rpsJ mutations in tetracycline resistance. Mutations located in the promoter region of patA also contributed to resistance to tetracycline, in addition to ciprofloxacin and ethidium bromide, by enhancing the transcription of the patA/B operon. Both mutants revealed a decreased accumulation of H3-tetracycline that was attributed to the increased expression of the PatA/B transporter. Mutations in the coding region of patA were also observed in both mutants and these contributed to resistance to tetracycline and ciprofloxacin, albeit to a lesser extent than the mutations in the promoter region. Comparative transcriptome profiling by RNA-sequencing further revealed a noticeable increase in the expression of several genes of the thiamine biosynthesis and salvage pathway in the two mutants and a role for some of these in resistance to tetracycline have been confirmed by gene inactivation. Conclusions The combination of genomic, transcriptomic and functional studies has allowed the discovery of novel tetracycline resistance mechanisms in S. pneumoniae.

背景 本研究旨在解析肺炎链球菌（Streptococcus pneumoniae）基因组固有四环素耐药性的决定因素。
结果 对两株四环素耐药且交叉耐药氟喹诺酮类环丙沙星（ciprofloxacin）的肺炎链球菌R6突变株开展全基因组测序，发现编码核糖体蛋白S10的rpsJ基因，以及编码ABC蛋白PatA与PatB的操纵子均发生突变。在四环素敏感的肺炎链球菌R6遗传背景中进行耐药性重构实验，证实了rpsJ突变在四环素耐药性形成中的关键作用。patA启动子区域的突变除可介导环丙沙星与溴化乙锭（ethidium bromide）的耐药性外，还可通过增强patA/B操纵子的转录，促进四环素耐药性的产生。两株突变株的H3-四环素蓄积量均出现下降，该现象归因于PatA/B转运蛋白表达水平的上调。两株突变株的patA编码区均存在突变，此类突变同样可介导四环素与环丙沙星耐药性，但其作用效果弱于启动子区域的突变。通过RNA测序（RNA-sequencing）开展的比较转录组分析进一步显示，两株突变株中硫胺素生物合成与补救合成通路的多个基因表达量显著上调；且通过基因失活实验已证实其中部分基因参与四环素耐药性的形成。
结论 本研究结合基因组学、转录组学与功能实验，成功发现了肺炎链球菌中新的四环素耐药机制。