Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Mechanism of action and limited cross-resistance of new lipopeptide MX-2401.

MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca²âº-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C55-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C55-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

The lipopeptide antibiotic Friulimicin B inhibits cell wall biosynthesis through complex formation with bactoprenol phosphate.

Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with an overall negative charge. Amphiphilicity is enhanced in the presence of Ca(2+), which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin, which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin to interrupt the cell wall precursor cycle through the formation of a Ca(2+)-dependent complex with the bactoprenol phosphate carrier C(55)-P, which is not targeted by any other antibiotic in use. Since C(55)-P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple pathways that are essential for a functional gram-positive cell envelope.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and reliable identification of clinical yeast isolates.

The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid routine identification of clinical yeast isolates. A total of 18 type collection strains and 267 recent clinical isolates of Candida (n = 250), Cryptococcus, Saccharomyces, Trichosporon, Geotrichum, Pichia, and Blastoschizomyces spp. were identified by MALDI-TOF MS. The results were compared with those obtained by conventional phenotyping and biochemical tests, including the API ID 32C system (bioMérieux, Nürtingen, Germany). Starting with cells from single colonies, accurate species identification by MALDI-TOF MS was achieved for 247 of the clinical isolates (92.5%). The remaining 20 isolates required complementation of the reference database with spectra for the appropriate reference strains which were obtained from type culture collections or identified by 26S rRNA gene sequencing. The absence of a suitable reference strain from the MALDI-TOF MS database was clearly indicated by log(score) values too low for the respective clinical isolates; i.e., no false-positive identifications occurred. After complementation of the database, all isolates were unambiguously identified. The established API ID 32C biochemical diagnostic system identified 244 isolates in the first round. Overall, MALDI-TOF MS proved a most rapid and reliable tool for the identification of yeasts and yeast-like fungi, with the method providing a combination of the lowest expenditure of consumables, easy interpretation of results, and a fast turnaround time.

Influence of Ca(2+) ions on the activity of lantibiotics containing a mersacidin-like lipid II binding motif.

Mersacidin binds to lipid II and thus blocks the transglycosylation step of the cell wall biosynthesis. Binding of lipid II involves a special motif, the so-called mersacidin-lipid II binding motif, which is conserved in a major subgroup of lantibiotics. We analyzed the role of Ca(2+) ions in the mode of action of mersacidin and some related peptides containing a mersacidin-like lipid II binding motif. We found that the stimulating effect of Ca(2+) ions on the antimicrobial activity known for mersacidin also applies to plantaricin C and lacticin 3147. Ca(2+) ions appear to facilitate the interaction of the lantibiotics with the bacterial membrane and with lipid II rather than being an essential part of a peptide-lipid II complex. In the case of lacticin 481, both the interaction with lipid II and the antimicrobial activity were Ca(2+) independent.

Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic.

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).

Analysis of in vitro activities and modes of action of synthetic antimicrobial peptides derived from an alpha-helical 'sequence template'.

OBJECTIVES: Cationic antimicrobial peptides (AMPs) are indispensable components of innate immune systems and promising candidates for novel anti-infective strategies. We rationally designed a series of peptides based on a template derived from known alpha-helical AMPs, which were then analysed regarding efficacy against clinical isolates and antibiotic mechanisms. METHODS: Efficacy tests included standard MIC and synergy assays. Whole cell assays with staphylococcal strains included killing kinetics, efflux experiments and determination of membrane depolarization. The transcriptional response of AMP-treated Staphylococcus aureus SG511 was analysed using a Scienion genomic microarray covering (approximately 90% of) the S. aureus N315 genome and AMP P16(6|E). RESULTS: The AMPs showed remarkable broad-spectrum activity against bacteria and fungi regardless of any pre-existing antibiotic resistance mechanism. Whole cell assays indicated that the AMPs target the cytoplasmic membrane; however, significant membrane leakage and depolarization was only observed with a standard laboratory test strain. Transcriptional profiling identified up-regulation of putative efflux pumps and of aerobic energy generation mechanisms as major counter activities. Important components of the staphylococcal cell wall stress stimulon were up-regulated and the lipid metabolism was also affected. CONCLUSIONS: The broad spectrum activity of amphiphilic helical AMPs is based on multiple stresses resulting from interactions with microbial membranes; however, rather than killing through formation of pores, the AMPs appear to interfere with the coordinated and highly dynamic functioning of membrane bound multienzyme complexes such as electron transport chains and cell wall or lipid biosynthesis machineries.

Molecular characterisation of aureocin A70, a multi-peptide bacteriocin isolated from Staphylococcus aureus.

Staphylococcus aureus A70 produces a heat-stable bacteriocin designated aureocin A70. Aureocin A70 is encoded within a mobilisable 8 kb plasmid, pRJ6, and is active against Listeria monocytogenes. Experiments of transposition mutagenesis and gene cloning had shown that aureocin A70 production and immunity were associated with the HindIII-A and B fragments of pRJ6. Therefore, a 6332 bp region of the plasmid, encompassing both these fragments, was sequenced using a concatenation DNA sequencing procedure. DNA sequence and genetic analyses revealed the presence of three transcriptional units that appear to be involved in bacteriocin activity. The first transcriptional unit contains a single gene, aurT, which encodes a protein that resembles an ATP-dependent transporter, similar to those involved in lantibiotic export. AurT is required for aureocin A70 production and it appears to be essential for mobilisation of pRJ6. The second putative operon contains two open reading frames (ORFs); the first gene, orfA, is predicted to encode a protein similar to small repressor proteins found in some Archaea, whose function remains to be elucidated. The second gene, orfB, codes for an 138 amino acid residue protein which shares a number of characteristics (high pI and hydrophobicity profile) with proteins associated with immunity, needed for self-protection against bacteriocin. Four other genes are present in the third operon, aurABCD. aurABCD encode four related peptides that are small (30-31 amino acid residues), strongly cationic (pI of 9.85 to 10.04) and highly hydrophobic. Theses peptides also have a high content of small amino acid residues like glycine and alanine, and no cysteine residue. Tn917-lac insertional mutations, which affected aureocin A70 activity, reside within operon aurABCD. Analysis of purified bacteriocin preparations by mass spectrometry demonstrated that all four peptides encoded by aurABCD operon are produced, expressed and excreted without post-translational modifications. Thus, aureocin A70 is a multi-peptide non-lantibiotic bacteriocin, which is transported without processing.

Unique peptide modifications involved in the biosynthesis of lantibiotics.

The lantibiotics are a unique class of bacterium-derived peptide antibiotics, all of which contain the rare amino acid lanthionine, as well as a number of other nonprotein amino acids. Unlike most other peptide antibiotics, lantibiotics are produced on the ribosome as a prepeptide, and a series of post-translational modifications converts this precursor into the biologically active peptide. A complex set of gene products involved in lantibiotic biosynthesis have been identified, including the genes for specific amino acid modifications, as well as other ancillary biosynthetic functions. In the future, it should be possible to use some of these enzymes to engineer novel, non-protein amino acids into other proteins of biotechnological interest and importance.

Interaction of the pore forming-peptide antibiotics Pep 5, nisin and subtilin with non-energized liposomes.

The cationic peptide antibiotics Pep 5, nisin and subtilin depolarize bacterial and artificial membranes by formation of voltage-dependent multi-state pores. Studies with non-energized liposomes indicated that the peptides do not span the membrane in the absence of a membrane potential. The effects of Pep 5 and nisin on neutral membranes, as studied by membrane fluidity, phase transition points and carboxyfluorescein efflux, were small compared to melittin. Acidic liposomes were affected more strongly, indicative of primarily electrostatic interactions with phospholipid head groups. Subtilin may slightly enter the hydrophobic core as suggested by tryptophan fluorescence quenching and liposome fusion experiments.

Multiple activities in lantibiotics--models for the design of novel antibiotics?

Lantibiotics are antibiotic peptides distinguished by the presence of the rare thioether amino acids lanthionine and/or methyllanthionine. They are produced by Gram-positive bacteria as gene-encoded precursor peptides and undergo post-translational modification to generate the mature peptide. The structural gene for the prepeptide and the genes involved in biosynthesis, processing, export as well as regulation and producer strain self-protection are organized in clusters. Based on their structural and functional features lantibiotics are currently divided into two major groups. The flexible amphiphilic type-A lantibiotics act primarily by pore formation in the bacterial membrane, a mechanism which was recently shown, e.g. for nisin and epidermin, to involve the interaction with specific docking molecules such as the membrane precursor lipid II. The rather rigid and globular type-B lantibiotics inhibit enzyme functions through interaction with the respective substrates: mersacidin and actagardine inhibit the cell wall biosynthesis by complexing lipid II, whereas the cinnamycin-like peptides inhibit phospholipases by binding phosphoethanolamine. Lantibiotics have attracted much attention in recent years and undergone extensive characterization. New insights into the mode of action and structure-function relationships as well as the biochemistry and the genetics will be outlined in this review.

Influence of cationic peptides on the activity of the autolytic endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22.

The peptidoglycan hydrolyzing endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22 is not able to attack intact cell walls of S. simulans 22, but hydrolyzes cell walls of Micrococcus luteus and soluble peptidoglycan chains of S. simulans 22. Hydrolysis of cell walls of M. luteus is activated in presence of organic cations such as poly-L-lysine (n = 17) and the peptide antibiotics Pep 5 and nisin, whereas hydrolysis of soluble peptidoglycan chains is not influenced. High concentrations of inorganic cations inhibit enzyme activity. These effects are discussed with respect to the cationic nature of the enzyme (pI greater than 9.5) and the regulation of the concerted action of the N-acetylmuramoyl-L-alanine amidase and the glucosaminidase during S. simulans 22 autolysis in vivo.

Cloning, sequencing and production of the lantibiotic mersacidin.

Mersacidin is a lanthionine-containing peptide antibiotic that shows a good in vivo efficiency against methicillin-resistant Staphylococcus aureus. It is excreted during early stationary phase and could be purified from culture supernatant in a one-step procedure by reversed phase HPLC. Its structural gene was cloned from chromosomal DNA of the producer strain Bacillus subtilis HIL Y-85,54728. Sequencing revealed that pre-mersacidin consists of an unusually long 48 amino acid leader sequence and a 20 amino acid propeptide part which is modified during biosynthesis to the mature lantibiotic. The comparison of the mersacidin prepeptide with those of hitherto known lantibiotics demonstrates that mersacidin is more closely related to type B lantibiotic cinnamycin than to type A lantibiotics.

Lantibiotic-mediated anti-lactobacillus activity of a vaginal Staphylococcus aureus isolate.

Staphylococcus aureus strain 26 inhibited the growth of 23 of 26 lactobacilli of endocervical origin, but only two of 17 staphylococci, in deferred antagonism tests. The inhibitory agent, a bacteriocin-like inhibitory substance (BLIS) named staphylococcin Au-26, was obtained from vigorously shaken liquid cultures containing a 0.1% (v/v) supplement of Tween 80 and was purified by chromatographic fractionation on XAD-2, carboxymethyl Sephadex and reversed phase HPLC. The molecular mass of staphylococcin Au-26 was estimated by SDS-PAGE to be approx. 2700. The detection of lanthionine residues in the molecule, the high stability to heating at acidic but not alkaline pH values and inactivation by proteinases indicate that staphylococcin Au-26 is a member of the lantibiotic class of peptide antibiotics--the first reported to be produced by a S. aureus strain. Primary sequence analysis showed that the N-terminus of the molecule is isoleucine, a characteristic also displayed by the lantibiotics nisin, epidermin and gallidermin.

Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis.

A gene (fur) for a Fur-like protein was identified on a 1.1 kb chromosomal DNA fragment of Staphylococcus epidermidis BN 280; the fur gene is followed by an open reading frame coding for the N-terminus of a putative superoxide dismutase. Within the -35 promoter region of both genes, as sequence motif was detected with low similarity to Fur-binding regulatory DNA segments, the so-called Fur boxes. Fur titration in Escherichia coli strain H1717 demonstrated that the E. coli Fur protein binds to the Fur box of the promoter region of the S. epidermidis fur gene. The S. epidermidis Fur protein was expressed in E. coli as indicated by the formation of inactive dimers with the chimeric repressor CI(N)-Fur(C) (Stojiljkovic, I. and Hantke. K. (1995) Mol. Gen. Genet. 247, 199-205), but was not able to complement the Fur mutation in E. coli H1681.

Effect of leader peptide mutations on biosynthesis of the lantibiotic Pep5.

Lantibiotics are lanthionine-containing antibiotic peptides which are synthesized from ribosomal prepeptides by post-translational modification. In order to elucidate the function of a conserved motif in the N-terminal leader sequence of lantibiotic prepeptides, three amino acids were exchanged in the leader peptide sequence of the lantibiotic Pep5. Exchanging Phe-19 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging Asp-6 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging ASp-6 for Lys, the production was decreased only to 82%. Proteolytic fragments of Pep5 or incorrectly modified Pep5 molecules, indicative of incorrect modifications, were not found in the culture supernatant. Thus, in contrast to the biosynthesis of the lantibiotic nisin, the FDLEI-motif is not essential for biosynthesis of Pep5 and has no influence on correct ring formation or processing, but seems to be important for optimal biosynthesis rates.

Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various gram-positive bacteria including Listeria sp.

This paper describes a simple purification method for the purification of carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola UI49. The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83. The protocol consists of only two purification steps, XAD chromatography and cation exchange chromatography. It is quick, easy, and can be used for large scale purification of these lantibiotics. The bactericidal activity of carnocin UI49 against carnobacteria, lactococci and Listeria was compared with that of nisin Z. The carnobacteria showed similar sensitivity towards carnocin UI49 and nisin. The nisin producing L. lactis strains were very sensitive towards carnocin UI49, while the non-producing L. lactis strains were more sensitive to nisin. The Listeria strains were weakly sensitive to carnocin UI49, lower concentrations of nisin were needed to inhibit growth.

GIS-supported investigation of a nosocomial Salmonella outbreak.

Within an outbreak at a university hospital 102 persons (44 patients, 26 nursery school children and one relative as well as 31 employees) have been diagnosed to be infected by Salmonella enteritidis. Ninety-nine persons complied with the "primary case"-definition. The source of infection could not be detected in retrospect by hygienic-microbiological methods due to missing food samples. But GIS (Geographical Information System)-supported epidemiological investigation and analysis of food production showed that most likely vanilla pudding had been the vehicle of infection. Contamination of the pudding could be put down to the fact that its production took place in direct spatial and temporal association with the preparation of turkey. Probably further infections caused by this primary source were avoided by immediate measures. The making out of an HACCP-concept as well as structural and technical short-term redevelopment measures proved to be decisive factors to decrease the risk of further infections. From these experiences, some recommendations could be derived for the investigation of food-borne outbreaks in hospitals.

Lantibiotics: mode of action, biosynthesis and bioengineering.

Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues. The overwhelming majority of the lantibiotics shows antibacterial activity. Some lantibiotics, e.g. nisin, are characterized by a dual mode of action. These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis. The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane. Recent results show that complexing of lipid II is widespread among lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane. In the two-component system of lacticin 3147, the two functions are performed by the two different peptides. The genetic information for production of lantibiotics is organized in gene clusters which contain a structural gene (lanA) for the lantibiotic prepeptide. The modifications are introduced by one biosynthetic enzyme (LanM) or a combination of a dehydratase (LanB) and a cyclase (LanC). These enzymes have been in the focus of recent bioengineering studies: The structure of NisC has been resolved, the reaction mechanism of LctM was elucidated and the active site residues were characterized by mutagenesis studies. In vitro modification systems have successfully been used to introduce thioether rings into other biologically active peptides. Furthermore, variant lantibiotics with enhanced properties have been engineered and at least one promising new lantibiotic with strong activity against multiresistant pathogens has been described.

Naturally processed dermcidin-derived peptides do not permeabilize bacterial membranes and kill microorganisms irrespective of their charge.

Dermcidin (DCD) is a recently described antimicrobial peptide, which is constitutively expressed in eccrine sweat glands and transported via sweat to the epidermal surface. By postsecretory proteolytic processing in sweat the dermcidin protein gives rise to several truncated DCD peptides which differ in length and net charge. In order to understand the mechanism of antimicrobial activity, we analyzed the spectrum of activity of several naturally processed dermcidin-derived peptides, the secondary structure in different solvents, and the ability of these peptides to interact with or permeabilize the bacterial membrane. Interestingly, although all naturally processed DCD peptides can adopt an alpha-helical conformation in solvents, they have a diverse and partially overlapping spectrum of activity against gram-positive and gram-negative bacteria. This indicates that the net charge and the secondary structure of the peptides are not important for the toxic activity. Furthermore, using carboxyfluorescein-loaded liposomes, membrane permeability studies and electron microscopy we investigated whether DCD peptides are able to permeabilize bacterial membranes. The data convincingly show that irrespective of charge the different DCD peptides are not able to permeabilize bacterial membranes. However, bacterial mutants lacking specific cell envelope modifications exhibited different susceptibilities to killing by DCD peptides than wild-type bacterial strains. Finally, immunoelectron microscopy studies indicated that DCD peptides are able to bind to the bacterial surface; however, signs of membrane perturbation were not observed. These studies indicate that DCD peptides do not exert their activity by permeabilizing bacterial membranes.