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

Abstract

Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors

“…To exploit the cell-free, low-volume nature of INRI-seq, we globally analyzed the influence of an antisense PNA on translation. In line with in vivo data 27, 28 , we found that acpP -PNA is specific for its target, acpP . Downregulation of PNA off-targets that harbor mismatches in the complementary sequence requires substantially higher PNA concentrations.…”

“…Gratifyingly, a recent study investigating the effects of acpP -PNA on global transcript levels in a uropathogenic E. coli (UPEC) strain observed very similar off-target effects. Of the four off-targets identified by INRI-seq, gpmM and ugpQ were among the top regulated transcripts upon addition of acpP -PNA 27 . These findings should aid future PNA design by providing a framework to weigh the predicted specificity of PNAs according to their predicted off-targets.…”

“…Lastly, since Ribo-seq is performed on living cells, it can be difficult to dissect direct and indirect effects on translation. This is exemplified by antisense antibiotics 24, 25, 26 , whose import via carrier peptides broadly affects gene expression in addition to the desired antisense inhibition of the targeted gene of interest 27, 28 .…”

Design and off-target prediction for antisense oligomers targeting bacterial mRNAs with the MASON webserver

“…Recent reports using different E. coli strains showed that PNA-mRNA melting temperature (T m ) positively correlates with growth inhibition (Goltermann et al 2019) and is associated with in vitro translation inhibition of the target gene (Popella et al 2022). We therefore integrated T m predictions for PNA-RNA duplexes as a feature in MASON (see methods).…”

“…To test whether these results are specific to Salmonella , we applied the same bioinformatics analysis to a published transcriptomic data set in which UPEC were treated with 11 different PNAs targeting various essential genes (Popella et al 2022). The overall transcriptomic pattern closely resembles the data in the present study (Figure 3A, B).…”