Ribosome maturation by YbeY stabilises Type III secretion system transcripts in enterohaemorrhagic E. coli

Enterohaemorrhagic E. coli is a significant human pathogen that utilises a type 3 secretion system (T3S) to colonise the reservoir host, cattle, and humans. Transposon mutagenesis showed the ybeZYX-Int operon to be required for normal T3S levels. Deletion analyses identified the endoribonuclease ybeY, which was previously linked to 16S ribosomal RNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had additional pleiotropic effects, including temperature sensitivity and reduced growth. UV-crosslinking revealed specific binding of ybeY to the “neck” and “beak” regions of 16S ribosomal RNA, but identified no significant sRNA association. Sub-inhibitory concentrations of the translation initiation inhibitor kasugamycin provoked degradation of a polycistronic T3S mRNA. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ∆ybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting the priorities for protein production. UV-crosslinking analysis of RNA-protein interactions was performed as previously described (Tree et al. 2014) in biological duplicate with a control (untagged) sample. PCR amplified cDNA libraries were sequenced using an Illumina MiSeq platform. Data analysis were performed using the pyCRAC software package (Webb et al., 2014) as described in Sy et al. (2017). To identify statistically significant YbeY binding sites pyCalculateFDR was used on mapped reads with settings: 100nt flanking sequence for features (-r 100), p-value threshold of 0.05 (-m 0.05), minimum coverage of 10 reads (--min=10), and 500 iterations (--iterations=500). YbeY binding sites with FDR<0.05, recovered in both HTF tagged replicates, and absent from the untagged control for retained for further analysis.

肠出血性大肠杆菌（Enterohaemorrhagic E. coli, EHEC）是一类重要的人类致病菌，其通过三型分泌系统（type 3 secretion system, T3S）定殖储存宿主牛以及人类。转座子诱变实验表明，ybeZYX-Int操纵子对于维持正常的三型分泌系统水平不可或缺。缺失分析鉴定出核糖核酸内切酶YbeY，该蛋白此前被报道与16S核糖体RNA（16S ribosomal RNA）成熟及小RNA（small RNA, sRNA）功能相关。肠出血性大肠杆菌中YbeY的缺失会引发额外的多效性效应，包括温度敏感性与生长能力下降。紫外交联实验显示，YbeY可特异性结合16S核糖体RNA的“颈部”与“喙部”区域，但未检测到其与小RNA存在显著结合关联。亚抑制浓度的翻译起始抑制剂春日霉素（kasugamycin）可诱导多顺反子三型分泌系统mRNA的降解。本研究据此推断，三型分泌系统对起始核糖体的耗竭尤为敏感，这解释了ΔybeY菌株中三型分泌系统活性受抑制的现象。翻译能力受损的细胞中，辅助毒力转录本可能会被优先降解，这或反映了细胞内蛋白质合成的优先级调控机制。RNA-蛋白质相互作用的紫外交联分析实验参照此前报道（Tree等，2014）开展，设置生物学重复样本及未标记标签的对照样本。经PCR扩增的cDNA文库采用Illumina MiSeq测序平台进行测序。数据分析参照Sy等（2017）的方法，采用pyCRAC软件包（pyCRAC software package，Webb等，2014）完成。为鉴定具有统计学显著性的YbeY结合位点，本研究对比对后的测序reads使用pyCalculateFDR工具进行分析，参数设置如下：特征区域侧翼序列长度为100nt（-r 100）、p值阈值为0.05（-m 0.05）、最小覆盖度为10条reads（--min=10），迭代次数为500次（--iterations=500）。将错误发现率（false discovery rate, FDR）小于0.05、在两组HTF标记重复样本中均被富集且未在未标记对照样本中检出的YbeY结合位点保留，用于后续分析。