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...

The tyrocidines, antimicrobial cyclic decapeptides from Bacillus aneurinolyticus, have potent activity with drug/disinfectant potential, specifically against Listeria monocytogenes. The tyrocidine activity is dependent on an amphipathic balance. Structure-activity relationship (SAR) analysis combined with principal component analysis showed the best activity correlation with hydropathy and solvent accessible volume (hydrophobicity parameters), Mr and molecular volume (steric/size parameters), coupled with rigid sequence and charge prerequisites. For potent activity against L. monocytogenes strains, there is a prerequisite for a Tyr or Phe in the (W/F)(w/f)NQ(Y/F/W) sequence of the variable pentapeptide and ornithine (Orn, O) as cationic residue in the conserved V(K/O)LfP pentapeptide, particularly with Trp in the aromatic dipeptide moiety of the variable pentapeptide. The roles of Trp and Orn in the tyrocidines were confirmed with most active peptide, tyrocidine B (TrcB) containing Orn and a Trp-D-Phe in the aromatic dipeptide moiety. However, a novel analogue with a trimethylated ornithine and Phe-D-Phe showed an activity rivalling that of TrcB. Our results emphasized that activity is dictated by interplay between the character of the aromatic residues in the variable pentapeptide and the cationic residue. Any residue change resulting in tighter membrane/cell wall interaction is likely to trap tyrocidines and impede their mechanism of action.

Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by Brevibacillus parabrevis, showed potent activity against chloroquine sensitive (CQS) Plasmodium falciparum. The identity of the aromatic residues in the aromatic dipeptide unit in cyclo-(D-Phe1-Pro2-(Phe3/Trp3)-D-Phe4/D-Trp4)-Asn5-Gln6-(Tyr7/Phe7/Trp7)-Val8-(Orn9/Lys9)-Leu10 was proposed to have an important role in activity. CQS and resistant (CQR) P. falciparum strains were challenged with three representative cyclodecapeptides. Our results confirmed that cyclodecapeptides from tyrothricin had significantly higher antiplasmodial activity than the analogous gramicidin S, rivaling that of CQ. However, the previously hypothesized size and hydrophobicity dependent activity for these peptides did not hold true for P. falciparum strains, other than for the CQS 3D7 strain. The Tyr7 in tyrocidine A (TrcA) with Phe3-D-Phe4 seem to be related with loss in activity correlating with CQ antagonism and resistance, indicating a shared target and/or resistance mechanism in which the phenolic groups play a role. Phe7 in phenycidine A, the second peptide containing Phe3-D-Phe4, also showed CQ antagonism. Conversely, Trp7 in tryptocidine C (TpcC) with Trp3-D-Trp4 showed improved peptide selectivity and activity towards the more resistant strains, without overt antagonism towards CQ. However, TpcC lead to similar parasite stage inhibition and parasite morphology changes than previously observed for TrcA. The disorganization of chromatin packing and neutral lipid structures, combined with amorphous hemozoin crystals, could account for halted growth in late trophozoite/early schizont stage and the nanomolar non-lytic activity of these peptides. These targets related to CQ antagonism, changes in neural lipid distribution, leading to hemozoin malformation, indicate that the tyrothricin cyclodecapeptides and CQ share a target in the malaria parasite. The differing activities of these cyclic peptides towards CQS and CQR P. falciparum strains could be due to variable target interaction in multiple modes of activity. This indicated that the cyclodecapeptide activity and parasite resistance response depended on the aromatic residues in positions 3, 4 and 7. This new insight on these natural cyclic decapeptides could also benefit the design of unique small peptidomimetics in which activity and resistance can be modulated.