Elucidation of the Bridging Pattern of the Lantibiotic Pseudomycoicidin.

Lantibiotics are post-translationally modified antibiotic peptides with lanthionine thioether bridges that represent potential alternatives to conventional antibiotics. The lantibiotic pseudomycoicidin is produced by Bacillus pseudomycoides DSM 12442 and is effective against many Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. While prior work demonstrated that pseudomycoicidin possesses one disulfide bridge and four thioether bridges, the ring topology has so far remained unclear. Here, we analyzed several pseudomycoicidin analogues that are affected in ring formation via MALDI-TOF-MS and tandem mass spectrometry with regard to their dehydration and fragmentation patterns, respectively. As a result, we propose a bridging pattern involving Thr8 and Cys13, Thr10 and Cys16, Ser18 and Cys21, and Ser20 and Cys26, thus, forming two double ring systems. Additionally, we localized the disulfide bridge to connect Cys3 and Cys7 and, therefore, fully elucidated the bridging pattern of pseudomycoicidin.

Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin.

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective ß-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin.

Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo.

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

Discovery of the Cyclic Lipopeptide Gacamide A by Genome Mining and Repair of the Defective GacA Regulator in Pseudomonas fluorescens Pf0-1.

Genome mining of the Gram-negative bacterium Pseudomonas fluorescens Pf0-1 showed that the strain possesses a silent NRPS-based biosynthetic gene cluster encoding a new lipopeptide; its activation required the repair of the global regulator system. In this paper, we describe the genomics-driven discovery and characterization of the associated secondary metabolite gacamide A, a lipodepsipeptide that forms a new family of Pseudomonas lipopeptides. The compound has a moderate, narrow-spectrum antibiotic activity and facilitates bacterial surface motility.

Deficient Lipid A Remodeling by the arnB Gene Promotes Biofilm Formation in Antimicrobial Peptide Susceptible Pseudomonas aeruginosa.

Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm-embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal microscopy, atomic force microscopy, and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose to lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a biofilm that is more robust than that of the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge that promote the formation of biofilms. Moreover, when grown under Mg2+ limitation, both the wild type and the arnB mutant exhibited a reduction in the level of membrane-bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a trade-off between LPS remodeling and resistance mechanisms of biofilm formation.

Sulfide Protects Staphylococcus aureus from Aminoglycoside Antibiotics but Cannot Be Regarded as a General Defense Mechanism against Antibiotics.

Sulfide production has been proposed to be a universal defense mechanism against antibiotics in bacteria (K. Shatalin, E. Shatalina, A. Mironov, and E. Nudler, Science 334:986-990, 2011, doi:10.1126/science.1209855). To gain insight into the mechanism underlying sulfide protection, we systematically and comparatively addressed the interference of sulfide with antibiotic activity against Staphylococcus aureus, as a model organism. The impact of sulfide and sulfide precursors on the antibiotic susceptibility of S. aureus to the most important classes of antibiotics was analyzed using modified disk diffusion assays, killing kinetic assays, and drug uptake studies. In addition, sulfide production and the impact of exogenously added sulfide on the physiology of S. aureus were analyzed. Sulfide protection was found to be limited to aminoglycoside antibiotics, which are known to be taken up by bacterial cells in an energy-dependent process. The protective mechanism was found to rely on an inhibitory effect of sulfide on the bacterial respiratory chain, leading to reduced drug uptake. S. aureus was found to be incapable of producing substantial amounts of sulfide. We propose that bacterial sulfide production should not be regarded as a general defense mechanism against antibiotics, since (i) it is limited to aminoglycosides and (ii) production levels vary considerably among species and, as for S. aureus, may be too low for protection.

Detection of methicillin-resistant coagulase-negative staphylococci harboring the class A mec complex by MALDI-TOF mass spectrometry.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

Antimicrobial Dialkylresorcins from Marine-Derived Microorganisms: Insights into Their Mode of Action and Putative Ecological Relevance.

Zobellia galactanivorans has been reported as a seaweed-associated or marine-derived species with largely unknown secondary metabolites. The combination of bioinformatic analysis and MS- and bioactivity guided separation led to the isolation of a new antibiotically active dialkylresorcin from the marine bacterium Z. galactanivorans. The antibiotic profile of the new dialkylresorcin zobelliphol (1: ) was investigated and compared with related and naturally occurring dialkyresorcins (i.e., stemphol (2: ) and 4-butyl-3,5-dihydroxybenzoic acid (3: )) from the marine-derived fungus Stemphylium globuliferum. Bacterial reporter strain assays provided insights into the mode of action of this antibiotic compound class. We identified an interference with bacterial DNA biosynthesis for the dialkylresorcin derivative 1: . In addition, the putative biosynthetic gene cluster corresponding to production of 1: was identified and a biosynthetic hypothesis was deduced.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.

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.

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.

Biosynthetic Origin of the Antibiotic Pseudopyronines†A and B in Pseudomonas putida BW11M1.

Within the framework of our effort to discover new antibiotics from pseudomonads, pseudopyronines A and B were isolated from the plant-derived Pseudomonas putida BW11M1. Pseudopyronines are 3,6-dialkyl-4-hydroxy-2-pyrones and displayed high in vitro activities against several human pathogens, and in our hands also towards the plant pathogen Pseudomonas savastanoi. Here, the biosynthesis of pseudopyronine B was studied by a combination of feeding experiments with isotopically labeled precursors, genomic sequence analysis, and gene deletion experiments. The studies resulted in the deduction of all acetate units and revealed that the biosynthesis of these α-pyrones occurs with a single PpyS-homologous ketosynthase. It fuses, with some substrate flexibility, a 3-oxo-fatty acid and a further unbranched saturated fatty acid, both of medium chain-length and provided by primary metabolism.

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides.

Lantibiotics are ribosomally synthesized antimicrobial peptides with substantial posttranslational modifications. They are characterized by the unique amino acids lanthionine and methyllanthionine, which are introduced by dehydration of Ser/Thr residues and linkage of the resulting dehydrated amino acids with Cys residues. BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database revealed a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Production of an antimicrobial substance with activity against Gram-positive bacteria was detectable in a cell wash extract of this strain. The substance was partially purified, and mass spectrometric analysis predicted a peptide of 2,786 Da in the active fraction. In order to characterize the putative lantibiotic further, heterologous expression of the predicted biosynthetic genes was performed in Escherichia coli. Coexpression of the prepeptide (PseA) along with the corresponding modification enzyme (PseM) resulted in the production of a modified peptide with the corresponding mass, carrying four out of eight possible dehydrations and supporting the presence of four thioether and one disulfide bridge. After the proteolytic removal of the leader, the core peptide exhibited antimicrobial activity. In conclusion, pseudomycoicidin is a novel lantibiotic with antimicrobial activity that was heterologously produced in E. coli.

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.

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.

Identification of agr-positive methicillin-resistant Staphylococcus aureus harbouring the class A mec complex by MALDI-TOF mass spectrometry.

A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411-2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.

Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ.

The worldwide spread of antibiotic-resistant bacteria has lent urgency to the search for antibiotics with new modes of action that are devoid of preexisting cross-resistances. We previously described a unique class of acyldepsipeptides (ADEPs) that exerts prominent antibacterial activity against Gram-positive pathogens including streptococci, enterococci, as well as multidrug-resistant Staphylococcus aureus. Here, we report that ADEP prevents cell division in Gram-positive bacteria and induces strong filamentation of rod-shaped Bacillus subtilis and swelling of coccoid S. aureus and Streptococcus pneumoniae. It emerged that ADEP treatment inhibits septum formation at the stage of Z-ring assembly, and that central cell division proteins delocalize from midcell positions. Using in vivo and in vitro studies, we show that the inhibition of Z-ring formation is a consequence of the proteolytic degradation of the essential cell division protein FtsZ. ADEP switches the bacterial ClpP peptidase from a regulated to an uncontrolled protease, and it turned out that FtsZ is particularly prone to degradation by the ADEP-ClpP complex. By preventing cell division, ADEP inhibits a vital cellular process of bacteria that is not targeted by any therapeutically applied antibiotic so far. Their unique multifaceted mechanism of action and antibacterial potency makes them promising lead structures for future antibiotic development.

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.

Model membrane studies for characterization of different antibiotic activities of lipopeptides from Pseudomonas.

Lipopeptides (LPs) are a structurally diverse class of amphipathic natural products that were in the past mainly known for their surfactant properties. However, the recent discovery of their antimicrobial and cytotoxic bioactivities have fueled and renewed the interest in this compound class. Propelled by the antimicrobial potential of this compound class, in this study a range of six underinvestigated LPs from Pseudomonads were examined with respect to their antibiotic activities towards bacteria. The assays revealed that only the glycosylated lipodipeptide SB-253514, produced by Pseudomonas strain SH-C52, showed significant antibacterial activity. Since the bioactivity of LPs is commonly attributed to membrane interactions, we analyzed the molecular interactions between the LPs and bacteria-like lipid model membranes in more detail via complementary biophysical approaches. Application of the quartz crystal microbalance (QCM) showed that all LPs possess a high binding affinity towards the model membranes. Despite their similar membrane affinity, monolayer studies displayed different tendencies of LPs to incorporate into the membrane. The degree of membrane incorporation could be correlated with specific structural features of the investigated LPs, such as distance between the peptidic macrocycle and the fatty acid, but did not fully reflect their respective antibacterial activity. Cyclic voltammetry (CV) experiments further demonstrated that SB-253514 showed no membrane permeabilization effects at inhibitory concentrations. Collectively, these results suggests that the antibacterial activity of SB-253514 cannot be explained by an unspecific detergent-like mechanism generally proposed for amphiphilic molecules but instead appears to occur via a defined structural target.

Antibiotic-induced autoactivation of IS256 in Staphylococcus aureus.

The 3' end of rsbU, encoding the positive regulator of the stress factor sigma B, was identified as a hot spot for spontaneous IS256 insertion in Staphylococcus aureus SA137/93G. Interestingly, subinhibitory concentrations of chloramphenicol in combination with heat stress, as well as linezolid and spectinomycin at physiological temperatures, selected for such rsbU::IS256 insertion mutants. In consequence of the inactivation of rsbU, the IS256 transposition frequency was increased 4-fold in S. aureus HG001.