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

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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.