A Novel Peptide Antibiotic Produced by Streptomyces roseoflavus Strain INA-Ac-5812 With Directed Activity Against Gram-Positive Bacteria

Abstract

In this work, we report the isolation and detailed functional characterization for the new non-ribosomally synthesized antibiotic 5812-A/C, which was derived from metabolites of Streptomyces roseoflavus INA-Ac-5812. According to its chemical structure, the studied 5812-A/C preliminary is composed of a cyclic peptide part covalently bounded with an arabinose residue. N-terminal amino acid sequencing of the native peptide has identified its partial structure of Leu-Asp-Gly-Ser-Gly and consisting of a Tyr residue that is supposed to have a two-component peptide nature for the molecule studied. However, the structural analysis of the antibiotic complex derived from S. roseoflavus INA-Ac-5812 is still ongoing. The mechanism of action of 5812-A/C was assessed in comparison with its most related analog, the lipopeptide antibiotic daptomycin, given the presence in both antimicrobials of an L-kynurenine amino acid residue. The inhibitory activity of 5812-A/C against Gram-positive bacteria including methicillin-resistant strain of Staphylococcus aureus was similar to daptomycin. The mechanism of action of 5812-A/C was associated with the disruption of membrane integrity, which differs in comparison with daptomycin and is most similar to the antimicrobial membrane-disturbing peptides. However, 5812-A/C demonstrated a calcium-dependent mode of action. In addition, unlike daptomycin, 5812-A/C was able to penetrate mature biofilms and inhibit the metabolic activity of embedded S. aureus cells. At the same time, 5812-A/C has no hemolytic activity toward erythrocyte, but possessed weak cytotoxic activity represented by heterochromatin condensation in human buccal epithelium cells. The biological properties of the peptide 5812-A/C suggest its classification as a calcium-dependent antibiotic effective against a wide spectrum of Gram-positive pathogenic bacteria.

Keywords: Streptomyces roseoflavus; anti-biofilm action; calcium-depending antibiotic; daptomycin; lipoglycopeptide antibiotic; peptide antibiotic.

FIGURE 1

Analytical reversed-phase HPLC analysis of INA-5812 antibiotic concentrate. (A) A profile is presented at 214 nm; (B) at 364 nm. Elution peak corresponding to 5812-A/C is marked by a black arrow.

FIGURE 2

The dependence of the inhibitory activity of antibiotics on the concentration of calcium in the medium. The MIC of 5812-A/C and daptomycin against S. aureus 209P was assessed at various concentrations of calcium, and the inhibition of 5812-A/C was found to be calcium-dependent.

FIGURE 3

Evaluation of membrane disturbance of S. aureus 209P using Live/Dead fluorescent dyes and agar-drop assay. (a,d,g,j) Intensity of SYTO 9 fluorescence. If bacterial membranes are permeabilized, propidium iodide penetrates into the cell. What follows is SYTO 9 getting displaced from nucleic acids, which leads to a decrease in fluorescence intensity in the green region (505–540 nm) of the spectrum. (b,e,h,k) Epifluorescence microscopy images of S. aureus 209P after 1 h of treatment. (c,f,i,l) Cell’s viability after exposure with antibiotics for 1 h. 5812-A/C (a–c), nisin (d–f), daptomycin (g–i), vancomycin (j–l). 1—The moment of introduction of the fluorophore; 2—the moment of introduction of the antibiotic. *Indicate significant differences at p < 0.05.

FIGURE 4

Antimicrobial activity of 5812-A/C against S. aureus 209P in preformed biofilms. Inhibition of the metabolic activity (TTC assay) of S. aureus cells, which are embedded in biofilm matrix compared with untreated samples. Treatment with: 5812-A/C (A), daptomycin (C), nisin (E); biofilm biomass (crystal violet assay) after the treatment with: 5812-A/C (B), daptomycin (D), nisin (F). *Indicate significant differences at p < 0.05.

FIGURE 5

Cytotoxic properties of 5812-A/C in relation to prokaryotic cells. Hemolytic activity of 5812-A/C, daptomycin, and indolicidin to human erythrocyte (A). Designations: NC—negative control (0.9% solution of NaCl); assay was repeated in triplicate, and percentage of hemolysis is expressed as mean ± SD. Influence of 5812-A/C on heterochromatin condensation located in the nuclei of human buccal epithelium cells (B).