Abstract
In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.
Highlights
The DNA sliding clamps are functionally and structurally conserved in all three domains of life, the sequence homologies are modest [1]
Whereas the PCNA interacting peptide (PIP)-box is found in many proteins essential for DNA replication, AlkB homolog PCNA Interacting Motif (APIM) is more frequently found in proteins important for handling of cellular stress, including proteins involved in DNA repair and translesion synthesis (TLS) [3,4,5,6,7,8,9]
It is well known that cationic peptides may have broad-spectrum antimicrobial activities, and this includes both synthetic peptides, such as cell penetrating peptides, and peptides synthesized by bacteria or eukaryotes [53]
Summary
The DNA sliding clamps are functionally and structurally conserved in all three domains of life, the sequence homologies are modest [1]. Two interacting motifs are identified for the trimeric eukaryote DNA sliding clamp PCNA; the PCNA interacting peptide (PIP)-box (Q-xx- -xx-) ( = aromatic, = hydrophobic, x = any residue) [2] and the AlkB homolog 2 PCNA Interacting Motif (APIM) (R/K- F/W/Y- L/I/V/A- L/I/V/A- K/R) [3]. APIM and PIP-box have overlapping interaction sites on PCNA that both include the conserved hydrophobic pocket below the interdomain connecting loop (IDCL) [10,11]. The -clamp is highly conserved between bacteria [13], and a loosely defined -clamp binding motif of 5–6 amino acids (QL-x1–2-LF) (CBM), found in all prokaryotic DNA polymerases [14,15], is suggested to be the prokaryotic ancestor to the PIP-box. CBM interacts with the hydrophobic pocket at the C-terminal part of the -clamp [16,17], similar to the APIM and PIP-box interactions with PCNA
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