A Staphylococcus capitis strain with unusual bacteriocin production.

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.

Thiosulfate transfer mediated by DsrE/TusA homologs from acidothermophilic sulfur-oxidizing archaeon Metallosphaera cuprina.

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S(0))- and tetrathionate (S4O6(2-))-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys(18)-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys(18)) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C(93)S and DsrE3A-C(101)S retained the ability to transfer the thiosulfonate group to TusA. TusA-C(18)S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.

Heterogeneity of host TLR2 stimulation by Staphylocoocus aureus isolates.

High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2) are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106). Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling.

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.

New proteins involved in sulfur trafficking in the cytoplasm of Allochromatium vinosum.

The formation of periplasmic sulfur globules is an intermediate step during the oxidation of reduced sulfur compounds in various sulfur-oxidizing microorganisms. The mechanism of how this sulfur is activated and crosses the cytoplasmic membrane for further oxidation to sulfite by the dissimilatory reductase DsrAB is incompletely understood, but it has been well documented that the pathway involves sulfur trafficking mediated by sulfur-carrying proteins. So far sulfur transfer from DsrEFH to DsrC has been established. Persulfurated DsrC very probably serves as a direct substrate for DsrAB. Here, we introduce further important players in oxidative sulfur metabolism; the proteins Rhd_2599, TusA, and DsrE2 are strictly conserved in the Chromatiaceae, Chlorobiaceae, and Acidithiobacillaceae families of sulfur-oxidizing bacteria and are linked to genes encoding complexes involved in sulfur oxidation (Dsr or Hdr) in the latter two. Here we show via relative quantitative real-time PCR and microarray analysis an increase of mRNA levels under sulfur-oxidizing conditions for rhd_2599, tusA, and dsrE2 in Allochromatium vinosum. Transcriptomic patterns for the three genes match those of major genes for the sulfur-oxidizing machinery rather than those involved in biosynthesis of sulfur-containing biomolecules. TusA appears to be one of the major proteins in A. vinosum. A rhd_2599-tusA-dsrE2-deficient mutant strain, although not viable in liquid culture, was clearly sulfur oxidation negative upon growth on solid media containing sulfide. Rhd_2599, TusA, and DsrE2 bind sulfur atoms via conserved cysteine residues, and experimental evidence is provided for the transfer of sulfur between these proteins as well as to DsrEFH and DsrC.

Biosynthetic origin of the antibiotic cyclocarbamate brabantamide A (SB-253514) in plant-associated Pseudomonas.

Within the framework of our genome-based program to discover new antibiotic lipopeptides from Pseudomonads, brabantamides A-C were isolated from plant-associated Pseudomonas sp. SH-C52. Brabantamides A-C displayed moderate to high in vitro activities against Gram-positive bacterial pathogens. Their shared structure is unique in that they contain a 5,5-bicyclic carbamate scaffold. Here, the biosynthesis of brabantamide A (SB-253514) was studied by a combination of bioinformatics, feeding experiments with isotopically labelled precursors and in vivo and in vitro functional analysis of enzymes encoded in the biosynthetic pathway. The studies resulted in the deduction of all biosynthetic building blocks of brabantamide A and revealed an unusual feature of this metabolite: its biosynthesis occurs via an initially formed linear di-lipopeptide that is subsequently rearranged by a novel FAD-dependent Baeyer-Villiger monooxygenase.

Cytoplasmic sulfurtransferases in the purple sulfur bacterium Allochromatium vinosum: evidence for sulfur transfer from DsrEFH to DsrC.

While the importance of sulfur transfer reactions is well established for a number of biosynthetic pathways, evidence has only started to emerge that sulfurtransferases may also be major players in sulfur-based microbial energy metabolism. Among the first organisms studied in this regard is the phototrophic purple sulfur bacterium Allochromatium vinosum. During the oxidation of reduced sulfur species to sulfate this Gammaproteobacterium accumulates sulfur globules. Low molecular weight organic persulfides have been proposed as carrier molecules transferring sulfur from the periplasmic sulfur globules into the cytoplasm where it is further oxidized via the "Dsr" (dissimilatory sulfite reductase) proteins. We have suggested earlier that the heterohexameric protein DsrEFH is the direct or indirect acceptor for persulfidic sulfur imported into the cytoplasm. This proposal originated from the structural similarity of DsrEFH with the established sulfurtransferase TusBCD from E. coli. As part of a system for tRNA modification TusBCD transfers sulfur to TusE, a homolog of another crucial component of the A. vinosum Dsr system, namely DsrC. Here we show that neither DsrEFH nor DsrC have the ability to mobilize sulfane sulfur directly from low molecular weight thiols like thiosulfate or glutathione persulfide. However, we demonstrate that DsrEFH binds sulfur specifically to the conserved cysteine residue DsrE-Cys78 in vitro. Sulfur atoms bound to cysteines in DsrH and DsrF were not detected. DsrC was exclusively persulfurated at DsrC-Cys111 in the penultimate position of the protein. Most importantly, we show that persulfurated DsrEFH indeed serves as an effective sulfur donor for DsrC in vitro. The active site cysteines Cys78 of DsrE and Cys20 of DsrH furthermore proved to be essential for sulfur oxidation in vivo supporting the notion that DsrEFH and DsrC are part of a sulfur relay system that transfers sulfur from a persulfurated carrier molecule to the dissimilatory sulfite reductase DsrAB.

Phosphorylation of amyloid-ß peptide at serine 8 attenuates its clearance via insulin-degrading and angiotensin-converting enzymes.

Accumulation of amyloid-ß peptides (Aß) in the brain is a common pathological feature of Alzheimer disease (AD). Aggregates of Aß are neurotoxic and appear to be critically involved in the neurodegeneration during AD pathogenesis. Accumulation of Aß could be caused by increased production, as indicated by several mutations in the amyloid precursor protein or the γ-secretase components presenilin-1 and presenilin-2 that cause familial early-onset AD. However, recent data also indicate a decreased clearance rate of Aß in AD brains. We recently demonstrated that Aß undergoes phosphorylation by extracellular or cell surface-localized protein kinase A, leading to increased aggregation. Here, we provide evidence that phosphorylation of monomeric Aß at Ser-8 also decreases its clearance by microglial cells. By using mass spectrometry, we demonstrate that phosphorylation at Ser-8 inhibited the proteolytic degradation of monomeric Aß by the insulin-degrading enzyme, a major Aß-degrading enzyme released from microglial cells. Phosphorylation also decreased the degradation of Aß by the angiotensin-converting enzyme. In contrast, Aß degradation by plasmin was largely unaffected by phosphorylation. Thus, phosphorylation of Aß could play a dual role in Aß metabolism. It decreases its proteolytic clearance and also promotes its aggregation. The inhibition of extracellular Aß phosphorylation, stimulation of protease expression and/or their proteolytic activity could be explored to promote Aß degradation in AD therapy or prevention.

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 ß-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.

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42.

UNLABELLED: 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. METHODOLOGY/PRINCIPAL FINDINGS: The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens. CONCLUSIONS/SIGNIFICANCE: The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.