Membrane interaction of a new synthetic antimicrobial lipopeptide sp-85 with broad spectrum activity

Abstract

ARTICLE IN PRESS• Sp-85 is a new synthetic antibiotic lipopeptide with broad spectrum activity.• Pressure-area curves of mixed monolayers show non-ideal mixing.• Binding to anionic liposomes results in fusion and leakage of aqueous contents.• TEM images of antibiotic-exposed bacteria show damaged membranes.• A mechanism of action based on bacterial membrane destabilization is proposed.g r a p h i c a l a b s t r a c t a r t i c l e i n f o a b s t r a c tAntimicrobial peptides offer a new class of therapeutic agents to which bacteria may not be able to develop genetic resistance, since their main activity is in the lipid component of the bacterial cell membrane. We have developed a series of synthetic cationic cyclic lipopeptides based on natural polymyxin, and in this work we explore the interaction of sp-85, an analog that contains a C12 fatty acid at the N-terminus and two residues of arginine. This analog has been selected from its broad spectrum antibacterial activity in the micromolar range, and it has a disruptive action on the cytoplasmic membrane of bacteria, as demonstrated by TEM. In order to obtain information on the interaction of this analog with membrane lipids, we have obtained thermodynamic parameters from mixed monolayers prepared with POPG and POPE/POPG (molar ratio 6:4), as models of Gram positive and Gram negative bacteria, respectively. Langmuir-Blodgett films have been extracted on glass plates and observed by confocal microscopy, and images are consistent with a strong destabilizing effect on the membrane organization induced by sp-85. The effect of sp-85 on the membrane is confirmed with unilamelar lipid vesicles of the same composition, where biophysical experiments based on fluorescence are indicative of membrane fusion and permeabilization starting at very low concentrations of peptide and only if anionic lipids are present. Overall, results described here provide strong evidence that the mode of action of sp-85 is the alteration of the bacterial membrane permeability barrier.

Synthesis of Some Analogues of Polymyxin E1 Antibiotic Using Automated Solid Phase Peptide Synthesis Assisted With Microwave Irradiation

“…The only Dab residue involved in lactam cyclization was substituted with another natural amino acid, Lys. Indeed, previous study confirmed the importance of the presence of two Arg residues in favoring electrostatic interactions of polymyxin [9,10]. On the other hand, several works from our lab studied the synthesis of Lys analogues of different types of polymyxins [13,14].…”

“…For instance, the replacement of the 6-methylheptanoic fatty acid in Polymyxin B with aromatic Fmoc molecule resulted in analogues with high antibacterial activity and significant reduced toxicity [8]. Another study confirmed the importance of the presence of two Arg residues in favoring electrostatic interactions of polymyxin [9,10]. An interesting work showed that adding the sequence formyl-Met-Leu-Phe to the linear tripeptide tail (L-Dab 1 -L-Thr 2 -L-Dab 3 ) conferred chemotactic and opsonic activities upon the conjugates, which were 8 to 10 times less toxic than the parent polymyxin antibiotic [11].…”

“…An interesting work showed that adding the sequence formyl-Met-Leu-Phe to the linear tripeptide tail (L-Dab 1 -L-Thr 2 -L-Dab 3 ) conferred chemotactic and opsonic activities upon the conjugates, which were 8 to 10 times less toxic than the parent polymyxin antibiotic [11]. Finally, introduction of acyl derivatives to γ-Dab 1 and Dab 9 of polymyxin was investigated, where L-Arg-Dab 9 showed a ten folded improvement in the PD 50 against Pseudomonas infection, while Arg-Dab 1 and Nitro-L-Arg-Dab 9 analogues were active but less than the parent compound [12]. More specifically, several works on polymyxin from our lab have been published over the years.…”

Bacteriostatic effect of lipopeptides from Bacillus subtilis N-2 on Pseudomonas putida using soybean meal by solid-state fermentation

“…To the best of our knowledge, this is the first report showing that lipopeptides inhibited the growth of P. putida. Grau-Campistany et al (2015) provided strong evidence that lipopeptides altered the bacterial membrane permeability barrier. Furthermore, Greber et al (2019) reported that lipopeptides showed a significant effect on cell membrane permeability.…”

Antimicrobial Resistance and Virulence Common Mechanisms

“…Histidine metabolism is also found to be involved in biofilm formation and is confirmed by gene disruption (Cabral et al, 2011). Histidine kinase is an important signaling molecule in biofilm formation in gram-positive and negative bacteria (McLoon et al, 2011;Shemesh and Chai, 2013;Grau et al, 2015). In addition, several ABC transporter system proteins were significantly up-regulated in the tylosin inhibiting biofilm formation.…”