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

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. Bacterial overnight cultures were diluted 1:100 in fresh MHB and grown to an OD600 of 0.5. Subsequently, obtained cultures were again diluted 1:100 in fresh MHB to adjust a cell concentration of approximately 106 cfu/mL. After transferring 1.9 mL of the bacterial solution into 5 mL low-binding tubes (LABsolute), 100 µL of 20x PPNA stock solutions were added to reach a final concentration of 5 µM for all KFF-conjugated PNAs. In parallel, an equal amount of cells was treated with the respective volume of sterile nuclease-free water, which was used as solvent for the test compounds, and served as negative control. After incubating the samples for 15 min at 37 °C, RNAprotect Bacteria (Qiagen) was added according to the manufacturer’s instructions. Following a 10-min incubation, cells were pelleted at 4 °C and 21,100 xg for 20 min. The supernatant was discarded and pellets were either directly used or stored at – 20 °C (< 1 day) for subsequent bacterial RNA isolation.