Harald Labischinski

Proteomic Approach to Understanding Antibiotic Action

ABSTRACT We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Dysregulation of bacterial proteolytic machinery by a new class of antibiotics.

Here we show that a new class of antibiotics—acyldepsipeptides—has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.

Specific and Potent Inhibition of NAD+-dependent DNA Ligase by Pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gramnegative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 μm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low μg/ml range, whereas eukaryotic cells were not affected up to 60 μg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.

The Lipopeptide Antibiotic Friulimicin B Inhibits Cell Wall Biosynthesis through Complex Formation with Bactoprenol Phosphate

ABSTRACT Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with an overall negative charge. Amphiphilicity is enhanced in the presence of Ca2+, which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin, which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin to interrupt the cell wall precursor cycle through the formation of a Ca2+-dependent complex with the bactoprenol phosphate carrier C55-P, which is not targeted by any other antibiotic in use. Since C55-P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple pathways that are essential for a functional gram-positive cell envelope.

A Spectrum of Changes Occurs in Peptidoglycan Composition of Glycopeptide-Intermediate Clinical Staphylococcus aureus Isolates

ABSTRACT 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-intermediateS. 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 afemC 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.

Cell Wall Composition and Decreased Autolytic Activity and Lysostaphin Susceptibility of Glycopeptide-Intermediate Staphylococcus aureus

ABSTRACT The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

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 staphylococcalfem 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 thefem mutants revealed that surface proteins were linked to cross-bridges containing one, three, or five glycyl residues, but not to the ε-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.

Medicinal chemistry optimization of acyldepsipeptides of the enopeptin class antibiotics.

The therapy of life-threatening infections is significantly weakened by the global spread of antibiotic resistance. Among Gram-positive bacteria, the development of resistance of staphylococci, streptococci, and enterococci is of particular concern. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of complicated nosocomial infections, and its prevalence in hospitals has increased during the last decade. Furthermore, the frequent reliance on vancomycin as a last line of defense has led to a high level of vancomycin-resistant nosocomial isolates. Streptococcus pneumoniae is an important community-acquired respiratory tract pathogen, and a leading cause of morbidity and mortality. More than 30% of US isolates are penicillin-resistant (penicillin-resistant S. pneumoniae, PRSP) and many of these strains are also resistant towards a large number of other antibiotics. Novel antibacterial agents with unprecedented mechanisms of action, which are devoid of pre-existing cross-resistances, are therefore very necessary. Most of the currently marketed antibiotic classes originate from the secondary metabolism of bacteria or fungi, which emphasizes that natural products are a valuable source of novel antibacterial agents. Thus, depsipeptides of the enopeptin family are of interest. In 1982, the isolation of depsipeptide antibiotics A54556A and B (Scheme 1, 1 and 2, respectively), from a mixture of eight individual depsipeptidic factors (A– H), produced by aerobic fermentation of Streptomyces hawaiiensis (NRRL 15010) was described. Enopeptin A (3) and B (4) (Scheme 1) were isolated in 1991 from a culture broth of Streptomyces sp. RK-1051, found in a soil sample from Tsuruoka City, Japan. The enopeptin structure consists of a lactone core composed of five S-configured amino acids and a lipophilic acylated phenylalanine side chain attached to a serine nitrogen. This macrocyclic, peptidic structure made these acyldepsipeptides interesting candidates for total synthesis. The level of interest was further increased by mode of action studies with B. subtilis, demonstrating impaired bacterial cell division and induction of filamentation as the underlying causes of antibacterial activity (Figure 1). Applying reversed genomics technologies, Brçtz-Oesterhelt and co-workers were able to demonstrate that the lead structure 1 acts by binding to caseine lytic protease (ClpP), the core unit of a major bacterial–protease complex. In order to protect the bacterial cell from the destructive power of this universal protease, ClpP is tightly regulated and requires a ClpATPase, and often other accessory proteins for activation. Binding of 1 to ClpP eliminates the requirement of Clp-ATPases and other regulatory factors for proteolytic degradation. Therefore, uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death. With respect to drug discovery, natural acyldepsipeptides 1 and 2 displayed only limited in vitro activity against the Grampositive pathogen S. aureus (Table 1) and Gram-negative bacteria were not susceptible. Furthermore, 1 and 2 were not effective in standard mouse models of lethal bacterial infection, and their physicochemical and pharmacokinetic profile was dominated by poor aqueous solubility and high clearance. Thus, 1 and 2 were not favorable candidates for drug development. Finally, natural enopeptin acyldepsipeptide antibiotics 1 and 2 are challenging lead structures from a chemical and synthetic viewpoint. Several functional groups limited their stability: (a) the lactone core was readily hydrolyzed in basic and acidic aqueous media; (b) the acylated serine hydroxy group eliminated readily under non-aqueous basic conditions; (c) the conjugated triene was sensitive to temperature and light (cyclization and aromatization reactions) ; and (d) solubility was not sufficient for parenteral application. However, the novel target and the absence of cross-resistance to established antibiotics strongly encouraged the initiation of a medicinal chemistry program, the objective of which was to improve these deficiencies using a thorough understanding of the lead conformation, based on x-ray structure analysis of the synthetic congener 5 (Figure 2). Crystallization of 5 from toluene gave a solvate with two toluene molecules, whereas solvent free crystals were obtained from aqueous acetonitrile. In the case of the solvate crystal, a Scheme 1. Natural enopeptin depsipeptide antibiotics.

Daptomycin versus Friulimicin B: In-Depth Profiling of Bacillus subtilis Cell Envelope Stress Responses

ABSTRACT The related lipo(depsi)peptide antibiotics daptomycin and friulimicin B show great potential in the treatment of multiply resistant gram-positive pathogens. Applying genome-wide in-depth expression profiling, we compared the respective stress responses of Bacillus subtilis. Both antibiotics target envelope integrity, based on the strong induction of extracytoplasmic function σ factor-dependent gene expression. The cell envelope stress-sensing two-component system LiaRS is exclusively and strongly induced by daptomycin, indicative of different mechanisms of action in the two compounds.

In Vitro Spectrum of Activity of Finafloxacin, a Novel, pH-Activated Fluoroquinolone, under Standard and Acidic Conditions

ABSTRACT Finafloxacin is a novel fluoroquinolone that exhibits enhanced antibacterial activity under acidic conditions. The aim of this study was to define the in vitro pH-activity relationship. Finafloxacin exhibited optimal antibacterial activity between pH 5.0 and 6.0 at which MICs were 4- to 8-fold lower than those determined at neutral pH. These observations were then confirmed against a larger collection of bacteria. These data suggest that finafloxacin could potentially offer a therapeutic advantage within acidic foci of infection.