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H Labischinski

Publications and source records attributed to H Labischinski.

At least 19 recordsLinked to original sources
PubMed2001

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus.

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.

Amino Acid Sequence↗
PubMed2001

Solid- and solution-phase synthesis of vancomycin and vancomycin analogues with activity against vancomycin-resistant bacteria.

Vancomycin, the prototypical member of the glycopeptide family of antibiotics, is a clinically used antibiotic employed against a variety of drug-resistant bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA). The recent emergence of vancomycin resistance, viewed as a growing threat to public health, prompted us to initiate a program aimed at restoring the potency of this important antibiotic through chemical manipulation of the vancomycin structure. Herein, we describe the development of synthetic technology based on the design of a novel selenium safety catch linker, application of this technology to a solid-phase semisynthesis of vancomycin, and the solid- and solution-phase synthesis of vancomycin libraries. Biological evaluation of these compound libraries led to the identification of a number of in vitro highly potent antibacterial agents effective against vancomycin-resistant bacteria. In addition to aiding these investigations, the solid-phase chemistry described herein is expected to enhance the power of combinatorial chemistry and facilitate chemical biology and medicinal chemistry studies.

Anti-Bacterial Agents↗
PubMed2001

Synthesis and biological evaluation of vancomycin dimers with potent activity against vancomycin-resistant bacteria: target-accelerated combinatorial synthesis.

Based on the notion that dimerization and/or variation of amino acid 1 of vancomycin could potentially enhance biological activity, a series of synthetic and chemical biology studies were undertaken in order to discover potent antibacterial agents. Herein we describe two ligation methods (disulfide formation and olefin metathesis) for dimerizing vancomycin derivatives and applications of target-accelerated combinatorial synthesis (e.g. combinatorial synthesis in the presence of vancomycin's target Ac2-L-Lys-D-Ala-D-Ala) to generate libraries of vancomycin dimers. Screening of these compound libraries led to the identification of a number of highly potent antibiotics effective against vancomycin-suspectible, vancomycin-intermediate resistant and, most significantly, vancomycin-resistant bacteria.

Anti-Bacterial Agents↗
PubMed2001

Emergence, mechanism, and clinical implications of reduced glycopeptide susceptibility in Staphylococcus aureus.

In order to understand the mechanism(s) of the resistance/reduced susceptibility of Staphylococcus aureus to glycopeptide antibiotics, the current data on the modes of action of glycopeptides were reviewed. In addition, the different test systems for detecting vancomycin resistance and the clinical relevance of resistant Staphylococcus aureus were analyzed. Finally, strategies to prevent the nosocomial spread of these bacteria are presented, as are new therapeutic options.

Anti-Bacterial Agents↗
PubMed2001

New antibiotics.

Explore the source record for details and available documents.

Anti-Bacterial Agents↗
PubMed2001

A spectrum of changes occurs in peptidoglycan composition of glycopeptide-intermediate clinical Staphylococcus aureus isolates.

The mechanism of glycopeptide resistance in Staphylococcus aureus is not known with certainty. Because the target of vancomycin is the D-Ala-D-Ala terminus of the stem peptide of the peptidoglycan precursor, by subjecting muropeptides to reversed-phase high-performance liquid chromatography, we investigated peptidoglycan obtained from glycopeptide-intermediate S. aureus (GISA) isolates for changes in composition and evaluated whether any peptidoglycan structural change was a consistent feature of clinical GISA isolates. GISA isolates Mu50 and Mu3 from Japan had the large glutamate-containing monomeric peak demonstrated previously, although strain H1, a vancomycin-susceptible MRSA isolate from Japan that was clonally related to Mu3 and Mu50, and a femC mutant that we studied, did also. For the U.S. GISA isolates, strain NJ had a large monomeric peak with a retention time identical to that described for the glutamate-containing monomer in strains H1, Mu3, and Mu50. However, a much smaller corresponding peak was seen in GISA MI, and this peak was absent from both GISA PC and a recent GISA isolate obtained from an adult patient in Illinois (strain IL). These data suggest that a uniform alteration in peptidoglycan composition cannot be discerned among the GISA isolates and indicate that a single genetic or biochemical change is unlikely to account for the glycopeptide resistance phenotype in the clinical GISA isolates observed to date. Furthermore, a large monomeric glutamate-containing peak is not sufficient to confer the resistance phenotype.

Anti-Bacterial Agents↗
PubMed2001

Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria.

We deleted a subset of 27 open reading frames (ORFs) from Escherichia coli which encode previously uncharacterized, probably soluble gene products homologous to proteins from a broad spectrum of bacterial pathogens such as Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis and only distantly related to eukaryotic proteins. Six novel bacteria-specific genes essential for growth in complex medium could be identified through a combination of bioinformatics-based and experimental approaches. We also compared our data to published results of gene inactivation projects with Mycoplasma genitalium and Bacillus subtilis and looked for homologs in all known prokaryotic genomes. Such analyses highlight the enormous metabolic flexibility of prokaryotes. Six of 27 studied genes have been functionally characterized up to now, amongst these four of the essential genes. The gene products YgbP, YgbB and YchB are involved in the non-mevalonate pathway of isoprenoid biosynthesis. KdtB is characterized as the posphopantetheine adenylyltransferase CoaD. There are indications that the other two essential gene products YjeE and YqgF, which we have identified, also possess enzymatic functions. These findings demonstrate the potential of such proteins to be used in screening of large chemical libraries for inhibitors which could be further developed to novel broad-spectrum antibiotics.

Bacillus subtilis↗
PubMed2000

The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated beta-lactam resistance.

Penicillin resistance in pneumococci is mediated by modified penicillin-binding proteins (PBPs) that have decreased affinity to beta-lactams. In high-level penicillin-resistant transformants of the laboratory strain Streptococcus pneumoniae R6 containing various combinations of low-affinity PBPs, disruption of the fib locus results in a collapse of PBP-mediated resistance. In addition, crosslinked muropeptides are highly reduced. The fib operon consists of two genes, fibA and fibB, homologous to Staphylococcus aureus femA/B which are also required for expression of methicillin resistance in this organism. FibA and FibB belong to a family of proteins of Gram-positive bacteria involved in the formation of interpeptide bridges, thus representing interesting new targets for antimicrobial compounds for this group of pathogens.

Amino Acid Sequence↗
PubMed1999

The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation.

The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in Staphylococcus aureus peptidoglycan was found to be encoded by the essential gene fmhB. We have analyzed murein composition and structure synthesized when fmhB expression is reduced. The endogenous fmhB promoter was substituted with the xylose regulon from Staphylococcus xylosus, which allowed glucose-controlled repression of fmhB transcription. Repression of fmhB reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropeptide was confirmed by MS to be N-acetylglucosamine-(beta-1,4)-N-acetylmuramic acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala), proving that FmhB is involved in the attachment of the first glycine to the pentaglycine interpeptide. This interpeptide plays an important role in crosslinking and stability of the S. aureus cell wall, acts as an anchor for cell wall-associated proteins, determinants of pathogenicity, and is essential for the expression of methicillin resistance. Any shortening of the pentaglycine side chain reduces or even abolishes methicillin resistance, as occurred with fmhB repression. Because of its key role FmhB is a potential target for novel antibacterial agents that could control the threat of emerging multiresistant S. aureus.

Carbohydrate Sequence↗
PubMed1999

Characterization of fmtA, a gene that modulates the expression of methicillin resistance in Staphylococcus aureus.

FmtA is a factor which affects the methicillin resistance level in methicillin-resistant Staphylococcus aureus. Since FmtA has two of three conserved motifs which are typically found in penicillin-binding proteins (PBPs) and beta-lactamases, we investigated the penicillin-binding activity of recombinant FmtA and found no such activity. Immunoblotting analysis revealed that FmtA localizes in the membrane fraction. To investigate the function of FmtA, high-pressure liquid chromatography analysis of cell wall muropeptides was performed with an fmtA-inactivated mutant and its parent. The mutant showed a reduced cross-linking and partially reduced amidation of glutamate residues in the peptidoglycan of the mutant. The transcription of fmtA was dose dependently increased by the addition of beta-lactam antibiotics, fosfomycin, and bacitracin, while its transcription was not changed by the addition of vancomycin or tetracycline. These results reveal that Fmt is a membrane-located, non-penicillin-binding protein and that mutation of fmtA affects the cell wall structure, although its precise function is still unknown.

Anti-Bacterial Agents↗
PubMed1998

Anchor structure of staphylococcal surface proteins. III. Role of the FemA, FemB, and FemX factors in anchoring surface proteins to the bacterial cell wall.

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the pentaglycyl cross-bridge in the staphylococcal peptidoglycan. Here, we asked whether altered peptidoglycan cross-bridges interfere with the sorting reaction and investigated surface protein anchoring in staphylococcal fem mutants. S. aureus strains carrying mutations in the femA, femB, femAB, or the femAX genes synthesize altered cross-bridges, and each of these strains displayed decreased sorting activity. Characterization of cell wall anchor structures purified from the fem mutants revealed that surface proteins were linked to cross-bridges containing one, three, or five glycyl residues, but not to the epsilon-amino of lysyl in muropeptides without glycine. When tested in a femAB strain synthesizing cross-bridges with mono-, tri-, and pentaglycyl as well as tetraglycyl-monoseryl, surface proteins were found anchored mostly to the five-residue cross-bridges (pentaglycyl or tetraglycyl-monoseryl). Thus, although wild-type peptidoglycan appears to be the preferred substrate for the sorting reaction, altered cell wall cross-bridges can be linked to the COOH-terminal end of surface proteins.

Amino Acid Sequence↗
PubMed1998

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.

Chromatography, High Pressure Liquid↗
PubMed1998

Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50.

We have previously reported methicillin-resistant Staphylococcus aureus clinical strains, Mu50 and Mu3, representing two categories of vancomycin resistance: Mu50 representing vancomycin-resistant S. aureus (VRSA) with MICs > or = 8 mg/L, and Mu3 representing hetero-VRSA with MICs < or = 4 mg/L using standard MIC determination methods. The mechanisms of vancomycin resistance in these strains were investigated. These strains did not carry the enterococcal vancomycin-resistance genes, vanA, vanB, or vanC1-3, as tested by PCR using specific primers. However, both strains produced three to five times the amount of penicillin-binding proteins (PBPs) 2 and 2' when compared with vancomycin-susceptible S. aureus control strains with or without methicillin resistance; the amounts of PBP2 produced in Mu3 and Mu50 were comparable to those in the vancomycin-resistant S. aureus mutant strains selected in vitro. Incorporation of 14C-labelled Nacetyl-glucosamine into the cell was three to 20 times increased in Mu50 and Mu3, and release of the radioactive cell wall material was increased in Mu3 (and also in Mu50, though to a lesser extent), compared with control strains. The amounts of intracellular murein monomer precursor in these strains were three to eight times greater than those found in control strains. Transmission electron microscopy showed a doubling in the cell wall thickness in Mu50 compared with the control strains. Mu3 did not show obvious cell wall thickening. These data indicate that activated synthesis and an increased rate of cell wall turnover are common features of Mu3 and Mu50 and may be the prerequisite for the expression of vancomycin resistance in S. aureus.

Acetylglucosamine↗
PubMed1998

Increase in glutamine-non-amidated muropeptides in the peptidoglycan of vancomycin-resistant Staphylococcus aureus strain Mu50.

The peptidoglycan compositions of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (MIC 8 mg/L) and hetero-VRSA strain Mu3 (MIC 3 mg/L) were compared in order to understand the mechanism of vancomycin resistance. As compared with Mu3, the cell wall of Mu50 had increased amounts of glutamine-non-amidated muropeptides and decreased cross-linking of peptidoglycan with a greatly decreased dimer/monomer ratio of muropeptides. In agreement with this observation, the peptidoglycan of Mu50 bound 1.4 times more vancomycin than that of Mu3. The increase in non-amidated muropeptides and the reduced cross-linking of the cell-wall peptidoglycan may contribute to the vancomycin resistance by increasing the consumption of vancomycin by the pre-existing cell wall of Mu50 and reducing the amount of vancomycin reaching the cytoplasmic membrane where the vital targets of the antibiotic are situated.

Amino Acids↗
PubMed1998

Novel anti-staphylococcal targets and compounds.

Many antimicrobial drugs have become less effective at combating infectious diseases, and experts in the field are concerned about the possibility of a 'post-antibiotic era' for some clinically important pathogens, particularly staphylococci. In our hospitals, nosocomial infections due to vancomycin-resistant enterococci have emerged, and there are concerns that the same resistance pattern may evolve in methicillin-resistant Staphylococcus aureus (MRSA). Examples from three main areas addressed to prevent this scenario are discussed: (i) screening of isolated biochemical targets and intact bacteria using high-throughput screening technologies, (ii) modifying existing compound classes like quinolones and glycopeptides to create more powerful compounds overcoming pathogen resistance and (iii) introduction of completely new classes of antibiotics.

Journal Article↗
PubMed1998

[Mechanism of vancomycin resistance in MRSA strain Mu50].

The mechanism of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA) Mu50 was investigated. More than 3 times increase of the incorporation of 14C-GluNAc into the cell wall of Mu50 was observed compared to those of vancomycin-susceptible strains FDA209P, H-1, LR5P1. The amount of cytoplasmic murein-monomer precursor increased more than 3 times in Mu50 compared to those of control strains. There was an increased production of PBP1, PBP2, and PBP2', which were 1.51, 17.2, and 7.06 times greater, respectively, in Mu50 than those in H-1, and 2.38, 4.46, and 1.96 times greater respectively, than those in LR5P1. By transmission electromicrograph, it was shown that the cell wall of Mu50 was twice thicker than that of LR5P1. Increase of tightly-bound vancomycin to the cell wall fraction was observed in Mu50 when compared to those in FDA209P and H-1 strains. From these results, the increase of the vancomycin targets, free D-Ala-D-Ala residues in the cell wall, in number, due to the activated cell wall synthesis, and/or decrease of the cross-linkage of the cell wall was suggested to be the mechanism of vancomycin resistance in the Mu50 strain.

Anti-Bacterial Agents↗
PubMed1998

[Increase of non-amidated muropeptides in the cell wall of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50].

The mechanism of resistance was studied with vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50. It was demonstrated that the incorporation of 14C-N-acetylglucosamine into the cell wall of Mu50 was not suppressed in the presence of 8 microliters/ml of vacomycin, whereas it was completely suppressed in vancomycin-susceptible strains FDA209P and H-1. Increased binding of vancomycin to the wall of Mu50 was observed compared to the control strains: 1.7 x 10(16) (Mu50), 6.1 x 10(15) (209P), and 6.7 x 10(15) (H-1) vancomycin molecules/mg cell wall, respectively. Remarkable proportion of the cell-wall component muropeptides were non-amidated in the cell wall of Mu50. In concordance with this phenomena, peptidoglycan cross-linkage decreased strikingly in the Mu50 strain. Free D-Ala-D-Ala residues at the end of muropeptides in the pre-existing cell wall generated by decreased cross-linkage seems to account for increased vancomycin binding. The increase of vancomycin-resistance level is presumably caused by sequestration of vancomycin molecules from primary target point on cell membrane. It was considered that at least two phenotypic changes are required for the vancomycin resistance in the Mu50 strain. First, as we have described previously, is the activated cell wall synthesis, and second, the reduction of cross-linkage of peptidoglycan by production of non-amidated muropeptide precursors.

Acetylglucosamine↗