Purification and biological activity of natural variants synthesized by tridecaptin M gene cluster and in vitro drug-kinetics of this antibiotic class

Abstract

The flexibility of the adenylation domains of non-ribosomal peptide synthetases (NRPSs) to different substrates creates a diversity of structurally similar peptides. In the present study, we investigated the antimicrobial activity of different natural variants synthesized by tridecaptin M gene cluster and performed the in vitro drug kinetics on this class. The natural variants were isolated and characterized using MALDI-MS and tandem mass spectrometry. All the peptides were studied for their antimicrobial activity in different pathogens, including colistin-resistant bacteria, and for haemolytic activity. Furthermore, in vitro drug kinetics was performed with tridecaptin M (or M1, the major product of the gene cluster). The natural variants displayed a varying degree of bioactivity with M11 showing the most potent antibacterial activity (MIC, 1–8 µg/ml), even against A. baumannii and P. aeruginosa strains. The in vitro kinetic studies revealed that tridecaptin M at a concentration of 16 µg/ml eradicated the bacteria completely in high-density culture. The compound demonstrated desirable post-antibiotic effect after two-hour exposure at MIC concentration. We also observed the reversal of resistance to this class of antibiotics in the presence of carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Altogether, the study demonstrated that tridecaptins are an excellent drug candidate against drug-resistant Gram-negative bacteria. Future studies are required to design a superior tridecaptin by investigating the interactions of different natural variants with the target.

In Vitro Evaluation of Antimicrobial Peptide Tridecaptin M in Combination with Other Antibiotics against Multidrug Resistant Acinetobacter baumannii

“…Tridecaptin M was tested to assess the effect on the outer membrane of different Gram-negative bacteria. We previously showed that tridecaptin M has weak antimicrobial activity in A. baumannii and was not active against P. aeruginosa [ 39 ]. However, similar to previously reported results, we observed strong permeabilization of the outer membrane in a concentration-dependent manner in all the pathogens as denoted by the increase in fluorescence intensity of N -phenyl-1-naphthylamin (NPN) dye ( Figure 2 a–c).…”

“…Recently, unacylated tridecaptin A 1 (without the lipid moiety; Figure 1 ), though ineffective itself, was reported to potentiate (or reduce the minimum inhibitory concentration of) Gram-positive antibiotics in Gram-negative bacteria including Enterobacteriaceae and A. baumannii [ 38 ]. Tridecaptins have very low intrinsic activity against A. baumannii [ 36 , 37 ], but they could still permeabilize the outer membrane very efficiently [ 39 ]. The naturally occurring tridecaptins are active against Enterobacteriaceae but have not been investigated for their potential in combination therapy.…”

The tridecaptins: non-ribosomal peptides that selectively target Gram-negative bacteria

“…Uptake of N-(1-naphthyl)aniline (NPN) dye by various Gram-negative strains confirmed OM binding by both TriM 1 and M 11 , while DiSC 3 (5) membrane depolarization assays are said to align with previous findings of disruption of the proton motive force. 24,30 Although further mechanism of action studies are required to confirm the cellular interactions responsible for the antimicrobial activity of the TriM variants, this novel class of NRAP-further highlights the potential of the tridecaptins as novel therapeutics for the treatment of stubborn bacterial infections.…”

“…No clear structure-activity relationship could be drawn from the range of analogues isolated, except that there may exist a synergistic relationship between certain amino acids, such that substitutions may impede activity. 24 The antimicrobial activity across the TriM family (92-98) is comparable to tridecaptin A and its variants such that they are active against Gram-negative E. coli, A. baumannii and even colistin-resistant strains of K. pneumoniae. TriM 11 (98) displayed the most potent antimicrobial activity and was even found to be highly active (MIC = 4 μg mL −1 ) against the polymyxin-resistant bacteria, Proteus mirabilis and Serratia marcescens.…”

“…After the initial resurgence of the tridecaptins, many new studies have been performed, including on their structural elucidation and/or biosynthesis, 17,[22][23][24] structure-activity relationships (SAR), [25][26][27][28][29] mechanism of action, 30 synergy with and conjugation to other antibiotics 31,32 and as selective Gram-negative-staining agents. 33…”