Transcriptional response of Enterococcus faecali to the glycosylated bacteriocin glycocin F

Glycocin F (GccF) is a bacteriocin produced by Lactobacillus plantarum which causes susceptible cells to enter bacteriostasis within minutes of exposure. GccF is diglycosylated with two N-acetylglucosamine (GlcNAc) moieties and is active against strains of Lb. plantarum, as well as potential pathogens such as Enterococcus faecalis and E. faecium. Its mechanism of action, however, is unknown. To better understand how GccF inhibits growth, we carried out RNA sequencing (RNA-seq) on E. faecalis strain JH2-2 exposed to different concentrations of GccF for different lengths of time. Here we demonstrate that free GlcNAc both protects E. faecalis from the effects of GccF, as well as pre-sensitises it to GccF. Transcriptional analysis showed that exposure to free GlcNAc resulted in up-regulation of a GlcNAc-specific phosphotransferase system (PTS) transporter, which is a putative GccF receptor. Time course analysis showed over 100 genes were up- or down-regulated within 10 minutes of exposure, a number that increased to over 300 genes after 40 minutes. A large number of these genes were found to be regulated by the alternative sigma factor 54, which was shown to not be required for susceptibility to GccF. Interestingly, genes encoding the V-type ATPases and sodium:proton antiporters were found to be up-regulated at all times and all concentrations of GccF tested. This suggests an indirect response to GccF, possibly due to low-level permeabilization of the cell membrane. Enterococcus faecalis cells were exposed to the bacteriocin glycocin F at different concentrations and different times, and transcriptional response profiled usind RNA sequncing on an Illumina HiSeq 2000. Samples were carried out in triplicate, with the necessary untreated controls.

糖杆菌素F（Glycocin F, GccF）是植物乳杆菌（Lactobacillus plantarum）产生的一类细菌素，可使敏感细胞在接触后数分钟内进入抑菌状态。GccF经双糖基化修饰，带有两个N-乙酰葡糖胺（N-acetylglucosamine, GlcNAc）基团，对植物乳杆菌菌株以及粪肠球菌（Enterococcus faecalis）、鹑鸡肠球菌（E. faecium）等潜在病原菌均具有抗菌活性，但其具体作用机制至今尚未阐明。
为深入解析GccF的生长抑制机制，我们对经不同浓度GccF处理不同时长的粪肠球菌JH2-2菌株开展了RNA测序（RNA-seq）分析。本研究证实，游离态GlcNAc既可保护粪肠球菌免受GccF的杀伤，也可预先致敏该菌对GccF的敏感性。转录组分析显示，暴露于游离GlcNAc会诱导GlcNAc特异性磷酸转移酶系统（phosphotransferase system, PTS）转运体的表达上调，而该转运体被推测为GccF的受体。
时间进程分析表明，接触GccF后10分钟内即有超过100个基因出现表达上调或下调，处理40分钟后该差异表达基因数量增至300余个。其中大量差异表达基因受替代σ因子54调控，但后续实验证实该σ因子并非粪肠球菌对GccF易感表型所必需。值得注意的是，编码V型ATP酶与钠氢反向转运体的基因在所有测试浓度与处理时长下均呈现表达上调，这提示粪肠球菌对GccF存在间接应答反应，其诱因可能为细胞膜的轻度通透化。
本研究将粪肠球菌暴露于不同浓度、不同处理时长的糖杆菌素F，随后通过Illumina HiSeq 2000测序平台对其转录应答谱进行分析。所有实验均设置三次生物学重复，并配备相应的未处理对照组。