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：
10.1371/journal.ppat.1009194
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发表时间：
2021-01
期刊：
影响因子：
6.7
通讯作者：
Butler CS
中科院分区：
文献类型：
--
作者：
Wagley S;Morcrette H;Kovacs-Simon A;Yang ZR;Power A;Tennant RK;Love J;Murray N;Titball RW;Butler CS
活的但不可培养(VBNC)状态是一种条件，在这种状态下细菌细胞是活的和代谢活跃的，但对使用常规生长介质培养具有抵抗力。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的细胞分选来选择性地分离VBNC细胞，并根据它们的代谢活性、细胞形状和致病能力来区分亚群。我们的结果表明，副溶血性弧菌VBNC细胞的两个亚群(P1和P2)存在，并且可以长期处于VBNC状态。VBNC亚群P2在应激条件下具有较好的生存适合性，在良好条件下表现出100%的复活。对这些亚群的蛋白质组分析表明，P2的蛋白质组比P1的蛋白质组更接近于开始微宇宙培养(T0)。鉴定了在不同VBNC群体之间显著上调或下调的蛋白质，并将差异调控蛋白质划分为23个功能基团，其中大部分属于代谢功能类别。负责将乳酸转化为丙酮酸的乳酸脱氢酶(LldD)蛋白在VBNC细胞的所有亚群中都显著上调。乳酸脱氢酶(RIMD2210633：ΔlldD)基因的缺失导致细胞比野生型细胞更快地进入VBNC状态，向VBNC细胞中添加乳酸有助于其复苏并延长复苏窗口。在RIMD2210633：Δ菌株中加入丙酮酸，恢复了野生型VBNC的形成轮廓。本研究提示乳酸脱氢酶可能在调节VBNC状态中发挥作用。据报道，变形杆菌的成员在暴露于压力或不允许的生长条件下时，会采取生存策略并进入可存活但不可培养(VBNC)状态。这是一种在自然环境中被广泛采用的特征，以使细菌能够在恶劣的环境条件下长期生存。尽管VBNC状态在微生物学中很重要，但我们对VBNC细胞的分子组成知之甚少。在这项研究中，我们使用模式生物副溶血性弧菌，我们发现在VBNC状态下存在着不同的细菌亚群，这些细菌具有不同的复苏潜力和不同的蛋白质组谱。我们还发现，编码乳酸脱氢酶(LldD)的基因的缺失触发了细胞进入VBNC状态，并且向VBNC细胞中加入乳酸延长了它们的复苏潜力窗口。细菌在VBNC状态下生存的能力可能与它们克服氧化应激的能力有关。
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 subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their 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 may play a role in regulating the VBNC state. Members of the Proteobacteria are reported to adopt a survival strategy and enter a viable but non culturable (VBNC) state, when exposed to stressful or non-permissible growth conditions. This is a characteristic employed widely in the natural environment in order for bacteria to survive harsh environmental conditions over a long period. In spite of the importance of the VBNC state in microbiology, we know little about the molecular makeup of VBNC cells. In this study, using the model organism Vibrio parahaemolyticus, we resolved that distinct subpopulations of bacteria exist in the VBNC state and these have different resuscitation potentials and distinct proteomic profiles. We also discovered that deletion of the gene encoding the enzyme lactate dehydrogenase (lldD) triggered the cells to enter the VBNC state, and adding lactate to VBNC cells extended their resuscitation potential window. The ability for bacteria to survive in the VBNC state might be linked to their ability to overcome oxidative stress.
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