Activating the Cpx response induces tolerance to antisense PNA delivered by an arginine-rich peptide in Escherichia coli

Abstract

Cell-penetrating peptides (CPPs) are increasingly used for cellular drug delivery in both pro-and eukaryotic cells, and oligoarginines have attracted special attention. How arginine-rich CPPs translocate across the cell envelope, particularly for prokaryotes, is still unknown. Arginine-rich CPPs efficiently deliver antimicrobial peptide nucleic acid (PNA) to its intracellular mRNA target in bacteria. We show that resistance to PNA conjugated to an arginine-rich CPP in Escherichia coli requires multiple genetic modifications and is specific for the CPP part and not to the PNA part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx*), which decreased the cytoplasmic membrane potential. This indicates an indirect energy-dependent uptake mechanism for antimicrobials conjugated to arginine-rich CPPs. In agreement, cpx* mutants showed low-level resistance to aminoglycosides and an arginine-rich CPP conjugated to a peptide targeting the DNA sliding clamp, i.e., similar uptake in E. coli for these antimicrobial compounds.

Translocation of non-lytic antimicrobial peptides and bacteria penetrating peptides across the inner membrane of the bacterial envelope

“…2) (Frimodt-Møller et al 2021). In agreement, an activated Cpx-response also conferred a decreased susceptibility to aminoglycosides and another arginine-rich BPP (R 11 ) conjugated to a peptide targeting the DNA sliding clamp (Frimodt-Møller et al 2021).…”

“…This indicates an indirect autonomous but energy-dependent uptake mechanism, relying on a high membrane potential, for antimicrobials conjugated to such an arginine-rich BPP (Fig. 2) (Frimodt-Møller et al 2021). In agreement, an activated Cpx-response also conferred a decreased susceptibility to aminoglycosides and another arginine-rich BPP (R 11 ) conjugated to a peptide targeting the DNA sliding clamp (Frimodt-Møller et al 2021).…”

“…However, very recent results are beginning to shed new light on the SbmA independent, autonomous uptake of at least arginine-rich BPPs and AMPs. Here, an activated Cpx-response was shown to confer resistance to the (R-X-R) 4 -Ahx-β-alanine-PNA conjugate in E. coli (Frimodt-Møller et al 2021). The activated Cpx-response resulted in the downregulation of respiratory genes leading to a decreased cytoplasmic membrane potential.…”

“…Arginine containing BPPs usually have an autonomous uptake across the cytoplasmic membrane (see below), suggesting that (RFF) 3 R-Ahx-β-alanine might face similar biostability issues in the periplasm as (KFF) 3 K-eg1. Compared to the straightforward resistance development to (KFF) 3 K-eg1-PNA, through loss of SbmA (Ghosal et al 2013), resistance development to peptide-PNA translocating through an autonomous uptake in E. coli requires multiple mutations and are difficult to obtain (Frimodt-Møller et al 2021). Thus, emphasizing the importance of improving carrier stability when optimizing/designing BPPs for delivery of antimicrobial peptides in bacteria.…”

Broad-Spectrum Antibacterial Peptide Kills Extracellular and Intracellular Bacteria Without Affecting Epithelialization

“…However, other resistance mechanisms not affecting the target, but the import of the peptide and/or degradation of the peptide, could lead to higher tolerance to betatide. It has recently been shown that the activation of the Cpx-envelope stress response system in E. coli can increase the tolerance toward antibacterial peptides and peptide nucleic acids (PNAs) containing arginine-rich CPPs by inhibiting the uptake (Frimodt-Moller et al, 2021). Betatide showed a 2× increase in MIC in Cpx mutant E. coli cells which had a constitutive active Cpx response (Frimodt-Moller et al, 2021).…”