Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp

Abstract

The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p)ppGpp (de la Fuente-Núñez et al. (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p)ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p)ppGpp synthesis moderately sensitizes – rather than protects – E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the stringent response messenger (p)ppGpp.

Synthetic Peptides to Target Stringent Response-Controlled Virulence in a Pseudomonas aeruginosa Murine Cutaneous Infection Model

“…Thus, to our knowledge, only the peptides 1018 and DJK-5 have been shown to provide a possible therapeutic approach against Gram-positive and Gram-negative pathogens in vertebrate infection models ( Achtman et al, 2012 ; Mansour et al, 2016 ). Recently, the observation that 1018 targets the cellular stress response had been challenged ( Andresen et al, 2016 ). However, the methodological procedures described by Andresen et al (2016) have serious issues including the use of microtiter plate adherence assays and crystal violet staining (which does not discriminate between live cells and bacterial debris) to assess biofilm formation.…”

“…Recently, the observation that 1018 targets the cellular stress response had been challenged ( Andresen et al, 2016 ). However, the methodological procedures described by Andresen et al (2016) have serious issues including the use of microtiter plate adherence assays and crystal violet staining (which does not discriminate between live cells and bacterial debris) to assess biofilm formation. Crystal violet experiments show large variations and are quite poor for accurately assessing anti-biofilm activity, especially when set up as a MIC experiment where peptides are added to planktonic cells.…”

Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA -Dependent and relA -Independent Tolerance to β-Lactams

“…These molecules were suggested to act either via direct inhibition of RSHs, such as the (p)ppGpp analogue Relacin (21), or via catalytic hydrolysis of (p)ppGpp, such as antibiofilm peptide 1018 and its derivatives (22, 23). However, our follow-up studies have shown that neither Relacin nor peptide 1018 specifically inhibits the stringent response in live cells (24, 25). …”

Biofilm dispersal: multiple elaborate strategies for dissemination of bacteria with unique properties

“…In contrast, a dodecamer peptide termed 1018, by specifically marking (p)ppGpp for degradation, promotes dispersal of biofilm of a wide range of Gram‐positive and Gram‐negative species (de la Fuente‐Núñez et al ., ; Reffuveille et al ., ). However, these findings have been recently challenged by a study showing that the dispersal activity of peptide 1018 does not rely on the (p)ppGpp response, but rather on an unknown mechanism (Andresen et al ., ).…”