Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria

Abstract

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.

Mygalin: An Acylpolyamine With Bactericidal Activity

“…The incubation of Mygalin with LPS caused a reduction in the absorbance as a function of the increase in the LPS concentration, indicating an interaction between the molecules. Similar results were described with peptides incubated with LPS (Lakshminarayanan et al, 2016;Sinha et al, 2017). Our data suggest that Mygalin may interact FIGURE 6 | Escherichia coli filamentation assay as indicative of DNA inhibition by Mygalin.…”

“…The possible interaction of Mygalin with LPS showed that there was an interaction between these two molecules, partially explaining its action against Gram-negative bacteria. Other drugs, such as curcumin and antimicrobial peptides, have the same ability to interact with LPS, reducing their activity (Lakshminarayanan et al, 2016;Sinha et al, 2017). The presence of chelating metals can interfere with E. coli metabolism, leading to its death.…”

“…The plates containing the samples were prepared in triplicate, and light was excluded during the experiments. The blank was endotoxin-free water (Lakshminarayanan et al, 2016).…”

Membrane Active Antimicrobial Peptides: Translating Mechanistic Insights to Design

“…These methods include the time-consuming mutation based empirical method, statistically based bioinformatics methods, and more sophisticated mechanism based methods such as MD simulations and biophysical experiments. Recently, multi-disciplinary approaches combining computational predictions, biophysical characterizations and biological validations have proved to be more promising (Bai et al, 2012 ; Koh et al, 2015a ; Lakshminarayanan et al, 2016 ). This section discusses various experimental and computational methods that have been used for AMP design.…”

Decreased expression of microRNA‑30b promotes the development of pulpitis by upregulating the expression of interleukin‑6 receptor

“…Obligate anaerobes and facultative anaerobes are the primary pathogens that cause pulpitis (4). Following lysis, Gram-negative bacteria release bacterial endotoxin, which has strong cytotoxicity and antigenicity (5). Endotoxin may directly destroy the local tissues and induce an inflammatory reaction (6).…”