Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus

Abstract

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. INTRODUCTIONSince the advent of antimicrobial use, there has been a progressive increase in drug resistance toward conventional antibiotics. This has instigated the search for an alternative class of antimicrobial agents with novel mechanisms of action and rare resistance (Brown & Wright, 2005). Antimicrobial peptides are potential candidates with membrane-linked mechanisms of action as well as possible cellular targets (Brown & Wright, 2005). Their rapid membranolytic activity reduces the likelihood of resistant mutants developing. Furthermore, reduced toxicities of the antimicrobial peptides through greater selectivity toward the more negatively charged bacterial cell membrane allow them to discriminate between pathogen targets and the neutral membranes of plants and animals (Javadpour et al., 1996;Matsuzaki et al., 1991Matsuzaki et al., , 1995Qin et al., 2003). Consequently, antimicrobial peptides show potential in the development of therapeutic agents to treat resistant strains of pathogenic microorganisms or to serve as bio-pesticides and preservatives (Brul & Coote, 1999;Cleveland et al., 2001;Keymanesh et al., 2009).A major limitation to the large-scale use of antimicrobial peptides has been the cost and efficiency of their production (Bradshaw, 2003;Gordon et al., 2005;Marr et al., 2006;Yeaman & Yount, 2003). Automated chemical synthesis to produce antimicrobial peptides remains very costly (Hancock & Lehrer, 1998;Hancock & Sahl, 2006;Marr et al., 2006), while use of transgenic organisms fo...

Synergistic Activity of the Tyrocidines, Antimicrobial Cyclodecapeptides from Bacillus aneurinolyticus, with Amphotericin B and Caspofungin against Candida albicans Biofilms

“…The tyrocidines were purified and analyzed for purity as described previously. De novo sequencing using electrospray mass spectrometry by Vosloo et al (34) and Spathelf (35) confirmed the identities of the tyrocidines and analogues. All the tyrocidines (TrcA, TrcB, TrcC, TpcC, and PhcA) used in this study had purities of Ͼ90% according to ultraperformance liquid chromatography linked to high-resolution electrospray mass spectrometry (see Table S1 in the supplemental material).…”

“…The percent leakage [F(%)] after 1 min was calculated by the following formula 100(%) (I Ϫ I 0 )/(I 100% Ϫ I 0 ), where I is the intensity observed in the peptide solution, and I 0 and I 100% are the fluorescence intensities measured in the absence of peptide and in the presence of Triton X-100, respectively (41). (29) and the identities were confirmed by our group (34,35). c Detected monoisotopic M r of peptides determined from purified peptides using highresolution time of flight-electrospray mass spectrometry (TOF-ESMS) (refer to Table S1 in the supplemental material).…”

Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi

“…The five tyrocidines and analogues, as well as the Trc mixture used in this study were obtained from the same stocks used by Troskie et al [5] and had purities >90% according to ultraperformance liquid chromatography linked to high resolution electrospray mass spectrometry (UPLC-MS), as described previously [24]. Gramicidin S (97.5% purity according to manufacturers, 94% purity according to our UPLC-MS studies) was supplied by Sigma (St Louis, USA).…”

Modelling the variable incorporation of aromatic amino acids in the tyrocidines and analogous cyclodecapeptides

“…Vosloo et al () established that the natural production of different tyrocidine analogues could be manipulated through supplementation of the growth medium of Br. parabrevis with the two aromatic amino acids (Aaa) Phe and Trp, while supplementation with Tyr, Cys, Lys and Arg had no noteworthy influence on the production or peptide profile.…”