Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival
Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival
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DOI：
10.1101/2020.07.23.216283
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发表时间：
2020-07
期刊：
影响因子：
6.7
通讯作者：
S. Wagley;Helen Morcrette;A. Kovács-Simon;Z. Yang;A. Power;Richard K. Tennant;J. Love;Neil Murray;R. Titball;C. Butler
中科院分区：
文献类型：
--
作者：
S. Wagley;Helen Morcrette;A. Kovács-Simon;Z. Yang;A. Power;Richard K. Tennant;J. Love;Neil Murray;R. Titball;C. Butler
活的但不可培养（VBNC）状态是细菌细胞是活的且代谢活跃的，但对使用常规生长培养基的培养具有抗性的状态。我们研究了副溶血性弧菌形成VBNC细胞并随后复苏的能力。在实验室中控制VBNC细胞形成的能力使我们能够使用荧光激活细胞分选来选择性地分离VBNC细胞，并根据它们的代谢活性、细胞形状和在大蜡螟中引起疾病的能力来区分亚群。我们的研究结果表明，存在两个关键的副溶血性弧菌VBNC细胞亚群（P1和P2），并可以在VBNC状态下保持长时间的休眠。两个亚群在有利条件下表现出不同的复苏能力。这些亚群的蛋白质组学分析（在两个不同的时间点：VBNC后12天（T12）和50天（T50））也揭示了P2的蛋白质组比P1的蛋白质组更类似于起始微宇宙培养物（T0）的蛋白质组。确定了不同VBNC群体之间显著上调和下调的蛋白质，并将显著调节的蛋白质分配到23个功能组中，大多数包括在代谢功能类别中。乳酸脱氢酶（lldD）蛋白在VBNC细胞的所有亚群中显著上调，并且负责将乳酸转化为丙酮酸。乳酸脱氢酶（RIMD 2210633：ΔlldD）基因的缺失导致细胞比野生型显著更快地进入VBNC状态，并且向VBNC细胞外源性添加乳酸盐有助于复苏并延长复苏窗口。向RIMD 2210633：ΔlldD菌株中添加丙酮酸盐恢复了野生型VBNC形成特征。这项研究表明，乳酸脱氢酶在调节VBNC状态中起着假定的关键作用。
The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two key subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. The two subpopulations displayed different abilities for revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) has also revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. The proteins that were significantly up and down regulated between the different VBNC populations were determined and significantly regulated proteins were assigned into 23 functional groups, the majority being included in metabolism functional categories. A lactate dehydrogenase (lldD) protein was significantly upregulated in all subpopulations of VBNC cells and is responsible for converting lactate to pyruvate. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene causes the cells to enter the VBNC state significantly faster than the wild-type, and exogenously adding lactate to VBNC cells aided resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase plays a putative key role in regulating the VBNC state.