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.
Cell penetrating peptides (CPP) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. However; their mechanism of action, particularly for prokaryotes is still unknown. Arginine-rich CPPs (R-CPP) efficiently delivers the antimicrobial peptide nucleic acid (PNA) into bacteria. Here, we show that resistance to an R-CPP PNA conjugate in Escherichia coli requires multiple genetic modifications and is specific to R-CPP 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 and thereby indicates an indirectly energy dependent uptake mechanism. Interestingly, cpx* mutants also showed increased tolerance to aminoglycosides and R-CPP conjugated to a peptide targeting the DNA sliding clamp; i.e., similar uptake in E. coli for these antimicrobial compounds. We speculate that the cpx* phenotype could create an evolutionary opportunity to adapt and evolve in the presence of either compounds.Author summaryThe emergence of multidrug resistant bacteria is raising the need for new classes of antibiotics. Peptide nucleic acids (PNAs) may fill this requirement by their ability to block translation of essential mRNAs and hence inhibit growth. PNA needs conjugation to a delivery peptide (cell penetrating peptide; CPP) to enter the bacteria. Arginine-rich CPPs (CPPR) are receiving a lot of attention for use as delivery vessels. Here, we show, for the first time, CPPR-PNA resistance in Escherichia coli directed towards the delivery peptide. Consequently, resistance also applies to other antimicrobial compounds delivered by the same carrier. An integral part of CPPR resistance is due to a constitutive active Cpx-response system, which leads to a decreased electric potential (ΔΨ) across the inner membrane. The decreased ΔΨ is a result of down-regulation of two aerobic respiratory operons, namely NADH:ubiquinone oxidoreductase complex I and cytochrome bo3 ubiquinol oxidase. The decreased ΔΨ also led to increased tolerance to aminoglycosides. This shows that a (large) negative ΔΨ is important for providing sufficient free energy for membrane translocation of both CPPR and that the inner membrane is the main barrier for entry of both arginine-rich delivery peptides and aminoglycosides.
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