Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics

Abstract

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.

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

“…Our current mechanistic understanding of PNA function is mostly derived from studies involving a small number of PNA targets (1,(6)(7)(8)13,18,26,29,30,68). Here, we present a systematic investigation of the antibacterial activity and mode of action of eleven PPNAs targeting different essential genes in UPEC.…”

“…Total RNA was purified from bacterial pellets using the miRNeasy Mini kit (Qiagen) according to the protocol #3 described in Popella et al (26). Briefly, cells were resuspended in 0.5 mg/mL lysozyme (Roth) in TE buffer (pH 8.0) and incubated for 5 min.…”

“…However, growth inhibition does not provide information on wider PNA-triggered cellular responses. We therefore recently employed RNA-sequencing (RNA-seq) as a readout for how PNAs affect target mRNA level, and how PNA treatment globally reshapes the transcriptome in Salmonella (26). Although PNAs are designed to block translation of their target , there is growing evidence that PNAs also cause target mRNA depletion (18,26,27,(29)(30)(31).…”

“…One widely-used PNA target is the essential gene acpP , which is involved in fatty acid biosynthesis (25). PNA- mediated silencing of acpP mRNA, using concentrations similar to conventional antibiotics, causes strong bactericidal effects in several bacterial species, including E. coli and Salmonella enterica (6– 8,26,27). Although additional PNA targets that mediate bacterial growth inhibition have been identified, as reviewed elsewhere (4, 5), acpP is the most promising one due to its exceptional antibacterial activity.…”

Robust Acquisition of High Resolution Spatial Transcriptomes from Preserved Tissues with Immunofluorescence Based Laser Capture Microdissection

“…S2) and should be broadly effective against many different RNases (Berger and Birkenmeier 1979). Although these complexes have been used to preserve RNA in tissues in a few studies previously (Shieh et al 2018;Credle et al 2017;Lyubimova et al 2013), they have been largely superseded by the more potent recombinant RNA inhibitors in most other experiments (Shapiro 2001;Dickson et al 2005;Mita et al 2021;Popella et al 2021;Qian et al 2020;Arizti-Sanz et al 2020). Whether RVC could provide robust RNA protection following immunolabelling, or immuno-LCM, which typically require many hours to complete, has not been previously examined.…”