Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent

Bactericidal Activity of the Human Skin Fatty Acid cis-6-Hexadecanoic Acid on Staphylococcus aureus

Human skin fatty acids are a potent aspect of our innate defenses, giving surface protection against potentially invasive organisms. They provide an important parameter in determining the ecology of the skin microflora, and alterations can lead to increased colonization by pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new avenue of exploration in the generation of control measures against drug-resistant organisms. Despite their importance, the mechanism(s) whereby skin fatty acids kill bacteria has remained largely elusive. Here, we describe an analysis of the bactericidal effects of the major human skin fatty acid cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen S. aureus. Several C6H concentration-dependent mechanisms were found. At high concentrations, C6H swiftly kills cells associated with a general loss of membrane integrity. However, C6H still kills at lower concentrations, acting through disruption of the proton motive force, an increase in membrane fluidity, and its effects on electron transfer. The design of analogues with altered bactericidal effects has begun to determine the structural constraints on activity and paves the way for the rational design of new antistaphylococcal agents

Production of the novel two-peptide lantibiotic lichenicidin by Bacillus licheniformis DSM 13.

BACKGROUND: Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. CONCLUSIONS/SIGNIFICANCE: In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains

Ultrashort Peptide Bioconjugates Are Exclusively Antifungal Agents and Synergize with Cyclodextrin and Amphotericin B

Many natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active against Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans but not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, for C. albicans, selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with β-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was tested in vitro and in vivo using toxicity assays on erythrocytes, macrophages, and mice. In vitro cytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and no in vivo murine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity

Gene inactivation of the lantibiotic modifications enzyme LicM1 (A) and LicM2 (B) by homologous recombination.

<p>Gene inactivations of the modifications enzymes LicM1 and LicM2 were performed by plasmid integration using the recombinant plasmids pMADDelLicM1AC and pMADLicM2AC. The resulting insertion mutants <i>B. licheniformis</i> LicM1INT (A) and LicM2INT (B) harbour two copies of the <i>lanM</i> genes within the lantibiotic gene cluster: a copy that is transcribed but is truncated and a second copy that does not dispose of promoter, Shine Dalgarno sequence and start codon. Genes that derive from the plasmid pMADDelLicM1AC are marked in green colours and those deriving from pMADLicM2AC are marked in orange and red.</p

Co-incubation of the isopropanol cell wash extract and the culture supernatant.

<p>In agar well diffusion assays, the isopropanol extract (25 µl) showed an antimicrobial activity against <i>S. aureus</i> ATCC 33592 (blue bar) while the culture supernantant was inactive (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006788#pone-0006788-g003" target="_blank">Fig. 3</a>). In order to test protease stability of the isopropanol extract, 25 µl of extract were mixed with 25 µl of culture supernatant (blue-green patterned bar), incubated for 2 h and then tested by agar diffusion. The co-incubation of both extracts had no effect on the activity of the isopropanol extract, indicating that the antimicrobial substance is stable against the proteases excreted by the producer strain.</p

Investigations to the Antibacterial Mechanism of Action of Kendomycin.

The emergence of bacteria that are resistant to many currently used drugs emphasizes the need to discover and develop new antibiotics that are effective against such multi-resistant strains. Kendomycin is a novel polyketide that has a unique quinone methide ansa structure and various biological properties. This compound exhibits strong antibacterial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Despite the promise of kendomycinin in several therapeutic areas, its mode of action has yet to be identified

Amino acid alignments of two-peptide lantibiotics.

<p>(A) Amino acid sequence alignment of the LicA1 propeptide with the LanA1 propeptides of the two-peptide lantibiotics plantaricin (PlwA1, AAG02567), staphylococcin C55 (SacA1, BAB78438), lacticin 3147 (LtnA1, O87236), haloduracin (HalA1, BAB04173), BHT (BhtA1, AAZ76603) and Smb (SmbA1, BAD72777) and, for comparison, the propeptide of mersacidin (MrsA, Z47559). Amino acid identities of the LanA1 propeptides are highlighted in green (100%), pink (75%) and blue (35%). The thioether bridging pattern represents that of the Halα and Plwα peptide. (B) Amino acid sequence alignment of the LicA2 propeptide with the Lanβ/LanA2 propeptides of the two-peptide lantibiotics plantaricin (PlwA2, AAG02566), staphylococcin C55 (SacA2, BAB78439), lacticin 3147 (LtnA2, O87237), haloduracin (HalA2, BAB04172), BHT (BhtA2, AAZ76602) and Smb (SmbA2, BAD72776). Amino acid identities are highlighted in green (100%), pink (75%) and blue (35%). The thioether bridging pattern represents that of the Halβ and Plwβ peptides.</p

Stability assay: Treatment with proteases.

<p>No loss of both antimicrobial activities was detected after treatment with trypsin and chymotrypsin for 4 h for both extracts. However, incubation of the isopropanol cell wash extract (blue bar) with proteinase K and pronase E resulted in a total loss of activity against <i>M. luteus</i> while the antimicrobial activity of the culture supernant (green column) was unaffected by these proteases.</p