Following tyrothricin peptide production by Brevibacillus parabrevis with electrospray mass spectrometry.

The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe1-L-Pro2-L-(Phe3/Trp3)-D-(Phe4/Trp4)-L-Asn5-L-Gln6-L-(Tyr7/Phe7/Trp7)-L-Val8-L-(Orn9/Lys9)-L-Leu10], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A "peptidomics" approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe3-D-Phe4 aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp3-D-Trp4. For the other variable aromatic residue position, Tyr7 was preferentially incorporated above Trp7, with a minor incorporation of Phe7 over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn9 to Lys9 incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery.

Chitosan-Based Film of Tyrothricin for Enhanced Antimicrobial Activity against Common Skin Pathogens Including Staphylococcus aureus.

Chitosan-based film-forming gel is regarded as a promising vehicle for topical delivery of antimicrobial agents to skin wounds, since it protects from microbial infection and the cationic polymer itself possesses antibacterial activity. In this study, possible synergistic interaction against common skin pathogens between the cationic polymer and tyrothricin (TRC), a cyclic polypeptide antibiotic, was investigated, by determining the concentration to inhibit 90% of bacterial isolates (MIC). The addition of the polysaccharide to TRC dramatically reduced the MIC values of TRC by 1/33 and 1/4 against both methicillin-resistant and methicillinsusceptible Staphylococcus aureus, respectively. The synergism of TRC and chitosan combination against both strains was demonstrated by the checkerboard method, with a fractional inhibitory concentration index below 0.5. Moreover, co-treatment of TRC and chitosan exhibited antibacterial activity against Pseudomonas aeruginosa, due to the antibacterial activity of chitosan, whereas TRC itself did not inhibit the gram-negative bacterial growth. These findings suggested that the use of chitosan-based film for topical delivery of TRC could be an alternative to improve TRC antimicrobial activity against strains that are abundant in skin wounds.

Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus.

A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.