From non-inherited to heritable antimicrobial peptide resistance: priming, aging, tolerance and persistence

抗菌素耐药性进化是一个需要进化研究的问题，既要了解宿主-微生物相互作用，又要了解耐药性的进化。最近，人们已经清楚地认识到，在耐药性的进化之前，可能存在诸如细菌持久性和耐受性等非遗传抗性机制。这可以通过暴露于亚致死浓度的抗菌素引起。我们之前已经表明，先前短时间暴露于低浓度（启动）抗菌肽（AMPs），多细胞生物的古老武器，诱导细菌耐受性和持久性增加，从而增加耐药性进化。在这里，我们想解决三个目标。(1)通过实验分析amp启动介导的耐受性和持久性及其影响。(2)了解不同动力学下amp启动的作用：对持续增加的应激源浓度的反应是否与启动反应的机制相同？(3)探讨细胞年龄对amp引物性的影响。年老的细胞和年轻的细胞受到的刺激程度一样吗？目标1:AMPs引发细菌增加在致命amp暴露下的存活率。存活的细胞要么代谢活跃，以较慢的速度死亡，要么更持久，代谢不活跃。要理解这种双相反应，需要使用一种装置使细胞同步。确定细菌存活表型将有助于探索细胞何时变得耐药（可遗传）或耐受/持久（不可遗传）。这些同步细胞上的RNAseq将允许识别候选基因，从而可以进行功能分析。目标2:amp等宿主的诱导免疫应答几乎总是从零开始：在免疫损伤信号传导后，诱导免疫效应物的表达不断增加。我们在这里想研究细菌启动（表现为细菌存活率、持久性和耐受性的增加）是否也发生在以不断增加的压力为代表的不同应激源动力学下。我们将使用带有大肠杆菌的微流体装置来测试这个问题。目标3：所有细胞都会衰老，细菌细胞也一样。在这里，通过同步细胞，然后使用确定年龄的“婴儿”细胞，我们将研究细胞年龄是否与启动性和启动反应的持续时间有关。这将有可能结合细胞同步装置与微流体，允许跟踪单个细胞的命运。解决这三个目标将使我们了解抗菌肽诱导启动后的双相反应，增加耐受性和持久性。这将是必要的，在进化动力学下，从启动和导致抗菌素耐药性进化。

Antimicrobial resistance evolution is a problem that requires evolutionary research, both, to understand host-microbe interactions and the evolution of drug resistance. Recently it has become clear that the evolution of resistance can be preceded by non-inheritable resistance mechanisms such as bacterial persistence and tolerance. This can be elicited by the exposure to sublethal antimicrobial concentrations. We have previously shown that prior short exposure to low concentrations (priming) of antimicrobial peptides (AMPs), ancient weapons of multicellular organisms, induce increased bacterial tolerance and persistence resulting in increased resistance evolution. Here we want to address three goals. (1) Disentangle AMP-priming mediated tolerance and persistence and their consequences experimentally. (2) Understand the role of AMP-priming under different kinetics: is the response to a continuously increasing concentration of stressors mediated by the same mechanisms as the priming response? (3) To investigate the role of cell age on primability by AMPs. Do older cells get primed to the same degree as younger cells? Goal 1 Bacterial priming by AMPs increases survival under lethal AMP-exposure. The surviving cells are either metabolically active dying at a slower rate or more persistent and metabolically inactive. To understand this biphasic response requires to synchronise cells using a device. Determining bacterial survival phenotypes will allow to explore when cells become either resistant (heritable) or tolerant/persistent (non-heritable). RNAseq on these synchronised cells will allow to identify candidate genes that can be followed up with functional analysis. Goal 2 Inducible immune responses of hosts such as AMPs almost always start from zero: after signalling of an immune insult, the expression of inducible immune effectors is continuously increasing. We here want to study whether bacterial priming (as manifested by increases in bacterial survival, persistence and tolerance) also occurs under the different stressor kinetics represented by continuously increasing stress. We will test this question using a microfluidic setup with E. coli. Goal 3 All cells age, also bacterial cells. Here by synchronizing cells and then use ‘baby’ cells of defined age we will investigate if cell age is related to primability and the duration of the priming response. This will be possible combining a cell synchronizing device with a microfluidic that allows to track the fate of individual cells. Addressing these three goals will allow us to understand the biphasic response, increased tolerance and persistence, after antimicrobial peptide induced priming. This will be essential to under the evolutionary dynamics resulting from priming and leading to antimicrobial resistance evolution.