The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Friedrich Götz - One of the best experts on this subject based on the ideXlab platform.
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vrah is the third component of the staphylococcus aureus vradeh system involved in Gallidermin and daptomycin resistance and pathogenicity
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Peter Popella, Mulugeta Nega, Sophia Krauss, Patrick Ebner, Julia Deibert, Friedrich GötzAbstract:In bacteria, extracellular signals are transduced into the cell predominantly by two-component systems (TCSs) comprising a regulatory unit triggered by a specific signal. Some of the TCSs control executing units such as ABC transporters involved in antibiotic resistance. For instance, inStaphylococcus aureus, activation of BraSR leads to the upregulation ofvraDEexpression that encodes an ABC transporter playing a role in bacitracin and nisin resistance. In this study, we show that the small staphylococcal transmembrane protein VraH forms, together with VraDE, a three-component system. Although the expression ofvraHin the absence ofvraDEwas sufficient to mediate low-level resistance, only this VraDEH entity conferred high-level resistance against daptomycin and Gallidermin. In most staphylococcal genomes,vraHis located immediately downstream ofvraDE, forming an operon, whereas in some species it is localized differently. In an invertebrate infection model, VraDEH significantly enhancedS. aureuspathogenicity. In analogy to the TCS connectors, VraH can be regarded as an ABC connector that modulates the activity of ABC transporters involved in antibiotic resistance.
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Epidermin and Gallidermin: Staphylococcal lantibiotics.
International journal of medical microbiology : IJMM, 2013Co-Authors: Friedrich Götz, Silvana Perconti, Peter Popella, Rolf G. Werner, Martin SchlagAbstract:The Staphylococcus epidermidis derived epidermin was the first lantibiotic that has been shown to be ribosomally synthesized and posttranslationally modified. Together with Gallidermin, produced by Staphylococcus gallinarum, they belong to the large class of cationic antimicrobial peptides (CAMPs) that act against a broad spectrum of Gram-positive bacteria. Here we describe the genetic organization, biosynthesis and modification, excretion, extracellular activation of the modified pre-peptide by proteolytic processing, self-protection of the producer, gene regulation, structure, and the mode of action of Gallidermin and epidermin. We also address mechanisms of bacterial tolerance to these lantibiotics and other CAMPs. Particularly Gallidermin has a high potential for therapeutic application, as it is active against methicillin-resistant Staphylococcus aureus strains (MRSA) and as it is able to prevent biofilm formation at sublethal concentrations.
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Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci
2013Co-Authors: Silvia Herbert, Andreas Peschel, Agnieszka Bera, Christiane Nerz, Dirk Kraus, Christiane Goerke, Michael Meehl, Ambrose Cheung, Friedrich GötzAbstract:It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R 115 (LP9), polymyxin B, or Gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilmnegative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7 % decrease in the content of esterified D-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound D-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA oroatA/graRS double mutant
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Concentration-dependent inhibition of QS-regulated phenotypes in Gram-negative bacteria.
2013Co-Authors: Ya-yun Chu, Mulugeta Nega, Martina Wölfle, Laure Plener, Stephanie Grond, Kirsten Jung, Friedrich GötzAbstract:(A) Prodigiosin production in S. marcescens. Cells were grown in MB medium with serial dilutions of yayurea A or B at 28°C. Relative prodigiosin production was calculated as the ratio between prodigiosin content (absorbance at 534 nm) and cell density (absorbance at 600 nm). (B) Bioluminescence in V. harveyi. Cells were grown in marine broth with serial dilutions of the compounds at 28°C for 24 h. Relative luminescence units were normalized by the cell density. (C) Biofilm formation of P. aeruginosa. Cells were grown in LB with serial dilutions of yayurea A or B at 37°C for 24 h. (D) Biofilm formation of S. aureus. Cells were grown in TSB with serial dilutions of yayurea A, B, or Gallidermin (positive control) at 37°C for 24 h. Biofilm cell layer was visualized by crystal violet staining and measured at 590 nm. Microtiter plates presented are representative of at least three independent sets of experiments. Bars indicate standard deviation of the mean, SD.
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activity of Gallidermin on staphylococcus aureus and staphylococcus epidermidis biofilms
Antimicrobial Agents and Chemotherapy, 2012Co-Authors: Mulugeta Nega, Jongkon Saising, Linda Dube, Annekathrin Ziebandt, Supayang Piyawan Voravuthikunchai, Friedrich GötzAbstract:Due to their abilities to form strong biofilms, Staphylococcus aureus and Staphylococcus epidermidis are the most frequently isolated pathogens in persistent and chronic implant-associated infections. As biofilm-embedded bacteria are more resistant to antibiotics and the immune system, they are extremely difficult to treat. Therefore, biofilm-active antibiotics are a major challenge. Here we investigated the effect of the lantibiotic Gallidermin on two representative biofilm-forming staphylococcal species. Gallidermin inhibits not only the growth of staphylococci in a dose-dependent manner but also efficiently prevents biofilm formation by both species. The effect on biofilm might be due to repression of biofilm-related targets, such as ica (intercellular adhesin) and atl (major autolysin). However, Gallidermin's killing activity on 24-h and 5-day-old biofilms was significantly decreased. A subpopulation of 0.1 to 1.0% of cells survived, comprising “persister” cells of an unknown genetic and physiological state. Like many other antibiotics, Gallidermin showed only limited activity on cells within mature biofilms.
Andreas Peschel - One of the best experts on this subject based on the ideXlab platform.
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inhibition of the atp synthase eliminates the intrinsic resistance of staphylococcus aureus towards polymyxins
Mbio, 2017Co-Authors: Martin Vestergaard, Andreas Peschel, Katrine Nohrmeldgaard, Martin Saxtorph Bojer, Christina Nielsen, Rikke Louise Meyer, Christoph Slavetinsky, Hanne IngmerAbstract:Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, Gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available antibiotics. Polymyxins constitute an important class of antibiotics for treatment of infections caused by Gram-negative pathogens, whereas Gram-positive bacteria remain largely insusceptible towards class of antibiotics. Here we performed a whole-genome screen among nonessential genes for polymyxin intrinsic resistance determinants in Staphylococcus aureus We found that the ATP synthase is important for polymyxin susceptibility and that inhibition of the ATP synthase sensitizes S. aureus towards polymyxins. Our study provides novel insights into the mechanisms that limit polymyxin activity against S. aureus and provides valuable targets for inhibitors to potentially enable the use of polymyxins against S. aureus and other Gram-positive pathogens.
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Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins
American Society for Microbiology, 2017Co-Authors: Martin Vestergaard, Andreas Peschel, Martin Saxtorph Bojer, Rikke Louise Meyer, Christoph Slavetinsky, Hanne Ingmer, Katrine Nøhr-meldgaard, Christina Krogsgård Nielsen, Fernando Baquero, Gerald B. PierAbstract:Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, Gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus. Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics
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Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci
2013Co-Authors: Silvia Herbert, Andreas Peschel, Agnieszka Bera, Christiane Nerz, Dirk Kraus, Christiane Goerke, Michael Meehl, Ambrose Cheung, Friedrich GötzAbstract:It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R 115 (LP9), polymyxin B, or Gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilmnegative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7 % decrease in the content of esterified D-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound D-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA oroatA/graRS double mutant
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Alanyl-Phosphatidylglycerol and Lysyl-Phosphatidylglycerol Are Translocated by the Same MprF Flippases and Have Similar Capacities To Protect against the Antibiotic Daptomycin in Staphylococcus aureus
Antimicrobial agents and chemotherapy, 2012Co-Authors: Christoph J. Slavetinsky, Andreas Peschel, Christoph M. ErnstAbstract:The lysinylation of negatively charged phosphatidylglycerol by MprF proteins reduces the affinity of cationic antimicrobial peptides (CAMPs) for bacterial cytoplasmic membranes and reduces the susceptibility of several Gram-positive bacterial pathogens to CAMPs. MprF of Staphylococcus aureus encompasses a lysyl-phosphatidylglycerol (Lys-PG) synthase and a Lys-PG flippase domain. In contrast, Clostridium perfringens encodes two MprF homologs which specifically synthesize alanyl-phosphatidylglycerol (Ala-PG) or Lys-PG, while only the Lys-PG synthase is fused to a putative flippase domain. It remains unknown whether cationic Lys-PG and zwitterionic Ala-PG differ in their capacities to be translocated by MprF flippases and if both can reduce CAMP susceptibility in Gram-positive bacteria. By expressing the MprF proteins of C. perfringens in an S. aureus mprF deletion mutant, we found that both lipids can be efficiently produced in S. aureus. Simultaneous expression of the Lys-PG and Ala-PG synthases led to the production of both lipids and slightly increased the overall amounts of aminoacyl phospholipids. Ala-PG production by the corresponding C. perfringens enzyme did not affect susceptibility to CAMPs such as nisin and Gallidermin or to the CAMP-like antibiotic daptomycin. However, coexpression of the Ala-PG synthase with flippase domains of Lys-PG synthesizing MprF proteins led to a wild-type level of daptomycin susceptibility, indicating that Ala-PG can also protect bacterial membranes against daptomycin and suggesting that Lys-PG flippases can also translocate the related lipid Ala-PG. Thus, bacterial aminoacyl phospholipid flippases exhibit more relaxed substrate specificity and Ala-PG and Lys-PG are more similar in their capacities to modulate membrane functions than anticipated.
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highly efficient staphylococcus carnosus mutant selection system based on suicidal bacteriocin activation
Applied and Environmental Microbiology, 2012Co-Authors: Bernhard Krismer, F Gotz, Gunther Thumm, Mulugeta Nega, Andreas PeschelAbstract:Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tu3928, which produces the highly active lanthionine-containing bacteriocin Gallidermin (lantibiotic), a strain was created producing inactive preGallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas preGallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of preGallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new preGallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.
Sven Panke - One of the best experts on this subject based on the ideXlab platform.
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Development of a fermentation process based on a defined medium for the production of preGallidermin, a nontoxic precursor of the lantibiotic Gallidermin
Applied Microbiology and Biotechnology, 2010Co-Authors: Giovanni Medaglia, Sven PankeAbstract:In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum ΔP , which produces preGallidermin (PGDM), a nontoxic precursor of the lantibiotic Gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum ΔP . The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum ΔP was grown up to a biomass concentration of 67 g l^−1 and produced 1.95 g l^−1 PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based Gallidermin production processes (Kempf et al. 2000 ). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tü 3928 was only 0.08 mM.
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development of a fermentation process based on a defined medium for the production of preGallidermin a nontoxic precursor of the lantibiotic Gallidermin
Applied Microbiology and Biotechnology, 2010Co-Authors: Giovanni Medaglia, Sven PankeAbstract:In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum DeltaP, which produces preGallidermin (PGDM), a nontoxic precursor of the lantibiotic Gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum DeltaP. The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum DeltaP was grown up to a biomass concentration of 67 g l(-1) and produced 1.95 g l(-1) PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based Gallidermin production processes (Kempf et al. 2000). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tu 3928 was only 0.08 mM.
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development of a high cell density protocol for the production of preGallidermin a non toxic precursor of the lantibiotic Gallidermin
Journal of Biotechnology, 2010Co-Authors: Giovanni Medaglia, Giorgia Valsesia, Sven PankeAbstract:Gallidermin, produced by Staphylococcus gallinarum Tu 3928, is a type-A lantibiotic with potential for the treatment of multidrug-resistant infections from Gram-positive pathogens such as methicillin-resistant S. aureus. In order to eliminate product inhibition as a reason for so far very modest product titers in S. gallinarum cultivations, we recently developed a novel two-stage production strategy based on the production of a non-toxic Gallidermin precursor - preGallidermin - by an engineered strain and the subsequent conversion of the precursor to Gallidermin in a separate step. This directed our efforts to the identification and alleviation of cultivation constraints for the engineered strain in fed-batch cultivations based on complex media supplemented with carbon sources, reasoning that extending the biomass production phase would lead to an extended preGallidermin production and higher titers. Substantial accumulation of acetate occurred in fed-batch cultivations with either maltose or glycerol - but not succinate - as an additional carbon source. Reductions in feeding rate to limit acetate accumulation led in turn to increased product degradation. Based on these observations, we developed an optimized exponential feeding strategy that allowed the process to reach a biomass concentration of 120gL(-1) and a product concentration of 1.23gL(-1) preGallidermin, corresponding to 0.780gL(-1) mature Gallidermin, a 2.5-fold increase over previous processes.
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circumventing the effect of product toxicity development of a novel two stage production process for the lantibiotic Gallidermin
Applied and Environmental Microbiology, 2007Co-Authors: Giorgia Valsesia, Giovanni Medaglia, Martin Held, W Minas, Sven PankeAbstract:Lantibiotics such as Gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, Gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tu3928 mutant, whose gene for the extracellular preGallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified Gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated preGallidermins showed no toxicity against Staphylococcus gallinarum Tu3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while Gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of preGallidermins and Gallidermin produced by S. gallinarum Tu3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature Gallidermin. In nonoptimized fermentations, the gdmP mutant produced preGallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on Gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.
Hans-georg Sahl - One of the best experts on this subject based on the ideXlab platform.
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Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane
2016Co-Authors: Hans-georg Sahl, Julia E. BAbstract:Mersacidin, Gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacte-rial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermedi-ate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, Gallidermin, andmersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane. Over the last decades, bacteria have demonstrated their im-pressive ability to adapt to changing environmental condi-tions by rapidly developing and accumulating antibiotic resis-tances. Helped by an extensive use of antibiotics in health care an
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Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane
Antimicrobial agents and chemotherapy, 2012Co-Authors: Michaela Wenzel, Hans-georg Sahl, Bastian Kohl, Daniela Münch, Nadja Raatschen, H. Bauke Albada, Leendert W. Hamoen, Nils Metzler-nolte, Julia E. BandowAbstract:Mersacidin, Gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, Gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.
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interaction of type a lantibiotics with undecaprenol bound cell envelope precursors
Microbial Drug Resistance, 2012Co-Authors: Anna Muller, Hans-georg Sahl, Hannah Ulm, Katrin Rederchrist, Tanja SchneiderAbstract:Lantibiotics are a unique group within the antimicrobial peptides characterized by the presence of thioether amino acids (lanthionine and methyllanthionine). These peptides are produced by and primarily act on Gram-positive bacteria exerting multiple activities at the cytoplasmic membrane of susceptible strains. Previously, the cell wall precursor lipid II was identified as the molecular target for the prototype lantibiotic nisin. Binding and sequestration of lipid II blocks the incorporation of the central cell wall precursor into the growing peptidoglycan network, thereby inhibiting the formation of a functional cell wall. Additionally, nisin combines this activity with a unique target-mediated pore formation, using lipid II as a docking molecule. The interaction with the pyrophosphate moiety of lipid II is crucial for nisin binding. We show that, besides binding to lipid II, nisin interacts with the lipid intermediates lipid III (undecaprenol-pyrophosphate-N-acetyl-glucosamine) and lipid IV (undecaprenol-pyrophosphate-N-acetyl-glucosamine-N-acetyl-mannosamine) of the wall teichoic acid (WTA) biosynthesis pathway. Binding of nisin to the precursors was observed at a stoichiometry of 2:1. The specific interaction with WTA precursors further promoted target-mediated pore formation in artificial lipid bilayers. Specific interactions with lipid III and lipid IV could also be demonstrated for related type A lantibiotics, for example, Gallidermin, containing the conserved lipid-II-binding motif.
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Membrane Lipids Determine the Antibiotic Activity of the Lantibiotic Gallidermin
Journal of Membrane Biology, 2008Co-Authors: Katrin Christ, Saad Al-kaddah, Imke Wiedemann, Bernd Rattay, Hans-georg Sahl, Gerd BendasAbstract:Lantibiotics, a group of lanthionine-containing peptides, display their antibiotic activity by combining different killing mechanisms within one molecule. The prototype lantibiotic nisin was shown to possess both inhibition of peptidoglycan synthesis and pore formation in bacterial membranes by interacting with lipid II. Gallidermin, which shares the lipid II binding motif with nisin but has a shorter molecular length, differed from nisin in pore formation in several strains of bacteria. To simulate the mode of action, we applied cyclic voltammetry and quartz crystal microbalance to correlate pore formation with lipid II binding kinetics of Gallidermin in model membranes. The inability of Gallidermin to form pores in DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (C18/1) and DPoPC (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine) (C16/1) membranes was related to the membrane thickness. For a better simulation of bacterial membrane characteristics, two different phospholipids with branched fatty acids were incorporated into the DPoPC matrix. Phospholipids with methyl branches in the middle of the fatty acid chains favored a lipid II–independent DPoPC permeabilization by Gallidermin, while long-branched phospholipids in which the branch is placed near the hydrophilic region induced an identical lipid II–dependent pore formation of Gallidermin and nisin. Obviously, the branched lipids altered lipid packing and reduced the membrane thickness. Therefore, the duality of Gallidermin activity (pore formation and inhibition of the cell wall synthesis) seems to be balanced by the bacterial membrane composition.
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Insights into In Vivo Activities of Lantibiotics from Gallidermin and Epidermin Mode-of-Action Studies
Antimicrobial agents and chemotherapy, 2006Co-Authors: Raquel Regina Bonelli, Tanja Schneider, Hans-georg Sahl, Imke WiedemannAbstract:The activity of lanthionine-containing peptide antibiotics (lantibiotics) is based on different killing mechanisms which may be combined in one molecule. The prototype lantibiotic nisin inhibits peptidoglycan synthesis and forms pores through specific interaction with the cell wall precursor lipid II. Gallidermin and epidermin possess the same putative lipid II binding motif as nisin; however, both peptides are considerably shorter (22 amino acids, compared to 34 in nisin). We demonstrate that in model membranes, lipid II-mediated pore formation by Gallidermin depends on membrane thickness. With intact cells, pore formation was less pronounced than for nisin and occurred only in some strains. In Lactococcus lactis subsp. cremoris HP, Gallidermin was not able to release K+, and a mutant peptide, [A12L]Gallidermin, in which the ability to form pores was disrupted, was as potent as wild-type Gallidermin, indicating that pore formation does not contribute to killing. In contrast, nisin rapidly formed pores in the L. lactis strain; however, it was approximately 10-fold less effective in killing. The superior activity of Gallidermin in a cell wall biosynthesis assay may help to explain this high potency. Generally, it appears that the multiple activities of lantibiotics combine differently for individual target strains.
Giovanni Medaglia - One of the best experts on this subject based on the ideXlab platform.
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Development of a fermentation process based on a defined medium for the production of preGallidermin, a nontoxic precursor of the lantibiotic Gallidermin
Applied Microbiology and Biotechnology, 2010Co-Authors: Giovanni Medaglia, Sven PankeAbstract:In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum ΔP , which produces preGallidermin (PGDM), a nontoxic precursor of the lantibiotic Gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum ΔP . The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum ΔP was grown up to a biomass concentration of 67 g l^−1 and produced 1.95 g l^−1 PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based Gallidermin production processes (Kempf et al. 2000 ). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tü 3928 was only 0.08 mM.
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development of a fermentation process based on a defined medium for the production of preGallidermin a nontoxic precursor of the lantibiotic Gallidermin
Applied Microbiology and Biotechnology, 2010Co-Authors: Giovanni Medaglia, Sven PankeAbstract:In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum DeltaP, which produces preGallidermin (PGDM), a nontoxic precursor of the lantibiotic Gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum DeltaP. The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum DeltaP was grown up to a biomass concentration of 67 g l(-1) and produced 1.95 g l(-1) PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based Gallidermin production processes (Kempf et al. 2000). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tu 3928 was only 0.08 mM.
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development of a high cell density protocol for the production of preGallidermin a non toxic precursor of the lantibiotic Gallidermin
Journal of Biotechnology, 2010Co-Authors: Giovanni Medaglia, Giorgia Valsesia, Sven PankeAbstract:Gallidermin, produced by Staphylococcus gallinarum Tu 3928, is a type-A lantibiotic with potential for the treatment of multidrug-resistant infections from Gram-positive pathogens such as methicillin-resistant S. aureus. In order to eliminate product inhibition as a reason for so far very modest product titers in S. gallinarum cultivations, we recently developed a novel two-stage production strategy based on the production of a non-toxic Gallidermin precursor - preGallidermin - by an engineered strain and the subsequent conversion of the precursor to Gallidermin in a separate step. This directed our efforts to the identification and alleviation of cultivation constraints for the engineered strain in fed-batch cultivations based on complex media supplemented with carbon sources, reasoning that extending the biomass production phase would lead to an extended preGallidermin production and higher titers. Substantial accumulation of acetate occurred in fed-batch cultivations with either maltose or glycerol - but not succinate - as an additional carbon source. Reductions in feeding rate to limit acetate accumulation led in turn to increased product degradation. Based on these observations, we developed an optimized exponential feeding strategy that allowed the process to reach a biomass concentration of 120gL(-1) and a product concentration of 1.23gL(-1) preGallidermin, corresponding to 0.780gL(-1) mature Gallidermin, a 2.5-fold increase over previous processes.
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circumventing the effect of product toxicity development of a novel two stage production process for the lantibiotic Gallidermin
Applied and Environmental Microbiology, 2007Co-Authors: Giorgia Valsesia, Giovanni Medaglia, Martin Held, W Minas, Sven PankeAbstract:Lantibiotics such as Gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, Gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tu3928 mutant, whose gene for the extracellular preGallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified Gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated preGallidermins showed no toxicity against Staphylococcus gallinarum Tu3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while Gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of preGallidermins and Gallidermin produced by S. gallinarum Tu3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature Gallidermin. In nonoptimized fermentations, the gdmP mutant produced preGallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on Gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.
