Institute of Molecular Infection Biology (IMIB), University of Würzburg, D-97080 Würzburg, Germany.
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), D-97080 Würzburg, Germany.
Nucleic Acids Res. 2021 May 7;49(8):4705-4724. doi: 10.1093/nar/gkab242.
Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.
反义肽核酸（PNA）抑制必需基因的 mRNA 为重新利用我们对细菌调控 RNA 的了解，开发可编程的物种特异性抗生素提供了一种直接的方法。虽然有大量证据表明 PNA 的有效性，但它们的靶标选择性和对细菌生理学的影响知之甚少。此外，虽然抗菌 PNA 通常设计用于阻断 mRNA 翻译，但对靶标 mRNA 水平的影响尚未得到充分研究。在这里，我们开创了使用全局 RNA-seq 分析以全转录组的方式破译 PNA 活性的先河。我们发现，基于 PNA 的反义寡核苷酸缀合物在沙门氏菌中强烈降低了广泛使用的靶基因 acpP 的 mRNA 水平，而脱靶效应有限。对几种不同 PNA-载体肽的系统分析不仅显示出不同的杀菌效率，而且还激活了应激途径。特别是 KFF-、RXR-和 Tat-PNA 缀合物特别诱导 PhoP/Q 反应，而后两者还另外触发了几个不同的途径。我们表明，PhoP/Q 反应的组成性激活可能导致 Tat-PNA 耐药性，这说明了 RNA-seq 用于理解 PNA 抗菌活性的实用性。总之，我们的研究为设计和评估 PNA 抗菌药物建立了一个实验框架，以长期寻求使用这些药物来精确编辑微生物组。
