Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenicEscherichia 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 11 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 9mer PNAs are generally as effective in inhibiting bacterial growth 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.

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 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, Supplemental Fig.…”

“…5B, E). This is likely an underestimate of the total number of effective off-targets, as we have recently shown that targeting of mRNAs by PNAs does not necessarily lead to depletion of the transcript (Popella et al 2022). Low PNA-mRNA affinity appears to ameliorate off-target activity, as we found that requiring a predicted T m of >20 °C increased the fraction of depleted off-targets to 13.6% and 25.1%, respectively (Supplemental Fig.…”

“…4A, B, Supplemental Fig. S9, (Popella et al 2021(Popella et al , 2022). Of the 369 unique significantly depleted genes, we observed off-target sites in TIRs of 192 genes (Fig.…”

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

“…Yet, this property can also be exploited to discriminate between direct and indirect effects: for example, we show that acpP -PNA directly inhibits translation of acpP , but not of fabF (Figure 5A , B ). In contrast, in vivo the PNA influences the stability of the entire dicistronic acpP - fabF mRNA ( 27 , 28 ), which makes it harder to assess its direct effect on translation.…”

“…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 ( 24–26 ), whose import via carrier peptides broadly affects gene expression in addition to the desired antisense inhibition of the targeted gene of interest ( 27 , 28 ).…”