New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO2.
New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO2.
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关于高压 CO2 诱导形成存活但不可培养的大肠杆菌 O157:H7 的新见解
DOI：
10.1128/mbio.00961-16
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发表时间：
2016-08-30
期刊：
影响因子：
6.4
通讯作者：
Liao X
中科院分区：
文献类型：
--
作者：
Zhao F;Wang Y;An H;Hao Y;Hu X;Liao X
摘要利用RNA测序（RNA-Seq）转录组学和同量异序标签相对和绝对定量（iTRAQ）蛋白质组学方法研究了高压CO2（HPCD）诱导的活但不可培养（VBNC）大肠杆菌O157：H7的形成。分析表明，97个基因和56个蛋白质在VBNC状态进入后发生了显著变化。VBNC细胞中与膜转运、中枢代谢、DNA复制和细胞分裂相关的基因和蛋白表达下调。这导致低代谢活性，同时细胞分裂停滞，这可能与VBNC状态形成有关。通过转录抑制因子z2046和外膜蛋白ompF的同源表达证实细胞分裂抑制和外膜过表达参与VBNC状态的形成。在VBNC状态进入后，细胞中的丙酮酸催化剂从三羧酸（TCA）循环转向发酵途径;这导致低水平的ATP。打击低能量供应，ATP生产中的VBNC细胞的L-丝氨酸和L-苏氨酸的降解，增加AMP的产生，和增强的电子传递补偿。此外，耐受性的细胞相对于HPCD诱导的酸，氧化，和高CO2应力增强，促进生产的氨和NADPH，并通过减少CO2生产过程中VBNC状态的形成。VBNC细胞中大多数与致病相关的基因和蛋白表达下调。这将降低细胞的致病性，这通过粘附测定证实。总之，降低代谢活性，抑制细胞分裂，提高存活能力。coliO157：H7可能引起HPCD诱导的VBNC状态的形成。重要性大肠杆菌O157：H7与全球大规模食源性疫情有关。据报道，食物中存在的10个细胞就可能导致疾病。而在大肠杆菌中，只有0.73~1.5个可培养的E.在日本，腌鲑鱼卵中的O157：H7大肠杆菌细胞引起了感染。研究人员发现，E.大肠杆菌O157：H7活菌但不可培养（VBNC）状态是此次暴发的源头。到目前为止，VBNC态的形成机制还不是很清楚。在以前的研究中，我们证明了高压CO2（HPCD）可以诱导E。coli O157：H7转化为VBNC状态。本研究采用RNA-Seq转录组学分析结合iTRAQ蛋白质组学方法研究VBNC E. coliO157：H7诱导表达。最后，我们提出了HPCD诱导VBNC细胞形成的可能机制，为控制HPCD诱导VBNC进入状态提供了理论依据。大肠杆菌O157：H7与世界范围内的大规模食源性疫情有关。据报道，食物中存在的10个细胞就可能导致疾病。而在大肠杆菌中，只有0.73~1.5个可培养的E.在日本，腌鲑鱼卵中的O157：H7大肠杆菌细胞引起了感染。研究人员发现，E.大肠杆菌O157：H7活菌但不可培养（VBNC）状态是此次暴发的源头。到目前为止，VBNC态的形成机制还不是很清楚。在以前的研究中，我们证明了高压CO2（HPCD）可以诱导E。coli O157：H7转化为VBNC状态。本研究采用RNA-Seq转录组学分析结合iTRAQ蛋白质组学方法研究VBNC E. coliO157：H7诱导表达。最后，我们提出了HPCD诱导VBNC细胞形成的可能机制，为控制HPCD诱导VBNC进入状态提供了理论依据。
ABSTRACT The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. IMPORTANCE Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment. Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.