Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa

Abstract

Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

Stereorandomization as a Method to Probe Peptide Bioactivity

“… 12 − 15 Here we asked the question whether SPPS using racemic rather than single enantiomer amino acids to form stereorandomized ( sr ) peptides might provide new insights into the mechanism of action of peptides. As a case study, we focused on antimicrobial peptides (AMPs), 16 − 19 antimicrobial peptide dendrimers (AMPDs), 20 − 28 and polymyxin B, 29 which are all membrane-disruptive compounds active on Gram-negative bacteria including multidrug resistant strains.…”

Peptides and Dendrimers: How to Combat Viral and Bacterial Infections

“…Peptide dendrimers based on di-, tetra- and octavalent lysine cores bound with tetra-(RLYR) or octapeptides (RLYRKVYG) elicited a strong antibacterial activity against both Gram-positives and Gram-negatives, with a major increase observed against Gram-negative bacteria [ 133 ]. The in vitro activity of the third-generation antimicrobial peptide dendrimer containing the dipeptide sequences KL (G3KL) showed significant activity against A. baumannii with minimal hemolytic toxicity [ 134 , 135 ]. A higher antimicrobial potency was correlated with a higher charge density and branching, and to a higher lipophilicity of the residues located at the C-terminus.…”

Application of Dendrimers for Treating Parasitic Diseases

“…Nanomedicine is therefore one of the most outstanding dendrimer applications. For instance, they have been widely used as nanocarriers for drug delivery [ 39 , 40 , 41 , 42 , 43 , 44 ], as diagnostic [ 45 , 46 , 47 , 48 , 49 ] and antimicrobial agents [ 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. Advances in synthetic chemistry and characterization approaches have allowed the production of many different types of dendrimers with distinct functionalities along with their applications.…”