Trichlorination of a Teicoplanin-Type Glycopeptide Antibiotic by the Halogenase StaI Evades Resistance.

Glycopeptide antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram-positive pathogens. These antibiotics are specialized metabolites produced by several genera of actinomycete bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68-kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all necessary genes for the synthesis of A47934, including two predicted halogenase genes, staI and staK In this study, we report that only one of the halogenase genes, staI, is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for antibiotic activity, as assessed by binding affinity for the target N-acyl-d-Ala-d-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of the enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of antibiotic.

More than just recruitment: the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH.

Glycopeptide antibiotic biosynthesis involves a complex cascade of reactions centred on a non-ribosomal peptide synthetase and modifiying proteins acting in trans, such as Cytochrome P450 enzymes. These P450s are responsible for cyclisation of the peptide via cross-linking aromatic amino acid side chains, which are a hallmark of the glycopeptide antibiotics. Here, we analysed the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934. Our results demonstrate that the P450 StaH is recruited to the NRPS machinery through interaction with the X-domain present in the last A47934 NRPS module. We determined the crystal structure of StaH and showed that it is responsible for the first cyclisation in A47934 biosynthesis and additionally exhibits flexible substrate specificity. Our results further point out that the X-domain has an impact on the efficiency of the in vitro cyclisation reaction: hybrid PCP-X constructs obtained by domain exchange between A47934 and teicoplanin biosynthesis NRPS modules reveal that the X-domain from A47934 leads to decreased P450 activity and alternate stereochemical preference for the substrate peptide. We determined that a tight interaction between StaH and the A47934 X-domain correlates with decreased in vitro P450 activity: this highlights the need for glycopeptide antibiotic cyclisation to be a dynamic system, with an overly tight interaction interfering with substrate turnover in vitro.

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold.

Although glycopeptide antibiotics (GPAs), including vancomycin and teicoplanin, represent the most important class of anti-infective agents in the treatment of serious gram-positive bacterial infections, their usefulness is threatened by the emergence of resistant strains. GPAs are complex natural products consisting of a heptapeptide skeleton assembled via nonribosomal peptide synthesis and constrained through multiple crosslinks, with diversity resulting from enzymatic modifications by a variety of tailoring enzymes, which can be used to produce GPA analogues that could overcome antibiotic resistance. GPA-modifying sulfotransferases are promising tools for generating the unique derivatives. Despite significant sequence and structural similarities, these sulfotransferases modify distinct side chains on the GPA scaffold. To provide insight into the spatial diversity of modifications, we have determined the crystal structure of the ternary complex of bacterial sulfotransferase StaL with the cofactor product 3'-phosphoadenosine 5'-phosphate and desulfo-A47934 aglycone substrate. Desulfo-A47934 binds with the hydroxyl group on the 4-hydroxyphenylglycine in residue 1 directed toward the 3'-phosphoadenosine 5'-phosphate and hydrogen-bonded to the catalytic His67. Homodimeric StaL can accommodate GPA substrate in only one of the two active sites because of potential steric clashes. Importantly, the aglycone substrate demonstrates a flattened conformation, in contrast to the cup-shaped structures observed previously. Analysis of the conformations of this scaffold showed that despite the apparent rigidity due to crosslinking between the side chains, the aglycone scaffold displays substantial flexibility, important for enzymatic modifications by the GPA-tailoring enzymes. We also discuss the potential of using the current structural information in generating unique GPA derivatives.

Click reaction synthesis of carbohydrate derivatives from ristocetin aglycon with antibacterial and antiviral activity.

New sugar derivatives of ristocetin were prepared by copper-catalyzed 1,3-dipolar cycloaddition reaction using azido-ristocetin aglycon and various propargyl glycosides. Some of them were found to be active against gram-positive bacteria and showed favorable antiviral activity against the H1N1 subtype of influenza A virus.

The biosynthesis of teicoplanin-type glycopeptide antibiotics: assignment of p450 mono-oxygenases to side chain cyclizations of glycopeptide a47934.

Streptomyces toyocaensis produces A47934, a teicoplanin-like type-IV glycopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. A47934 differs from the type-I vancomycin glycopeptides, which possess a tricyclic peptide backbone, by the presence of an additional ring closure between the aromatic amino acids 1 and 3. To elucidate the order of crosslinking reactions, P450 mono-oxygenase-inactivation mutants (DeltastaF, DeltastaG, DeltastaH, and DeltastaJ) of the A47934 producer were generated, and the accumulated intermediates were analyzed. Thus, the formation of each crosslink could unambiguously be assigned to a specific oxygenase. The structure of the released intermediates from the wild-type nonribosomal peptide synthetase assembly line facilitated the determination of the cyclization order. Unexpectedly, the additional ring closure in A47934, catalyzed by StaG, is the second oxygenase reaction.

Crystal structure of StaL, a glycopeptide antibiotic sulfotransferase from Streptomyces toyocaensis.

Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vancomycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934, a unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys(12), His(67), and Ser(98)), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp(34), His(43), Phe(77), Trp(132), and Glu(205).

Comparative analysis and insights into the evolution of gene clusters for glycopeptide antibiotic biosynthesis.

The bal, cep, dbv, sta and tcp gene clusters specify the biosynthesis of the glycopeptide antibiotics balhimycin, chloroeremomycin, A40926, A47934 and teicoplanin, respectively. These structurally related compounds share a similar mechanism of action in their inhibition of bacterial cell wall formation. Comparative sequence analysis was performed on the five gene clusters. Extensive conserved synteny was observed between the bal and cep clusters, which direct the synthesis of very similar compounds but originate from two different species of the genus Amycolatopsis. All other cluster pairs show a limited degree of conserved synteny, involving biosynthetically functional gene cassettes: these include those involved in the synthesis of the carbon backbone of two non-proteinogenic amino acids; in the linkage of amino acids 1--3 and 4--7 in the heptapeptide; and in the formation of the aromatic cross-links. Furthermore, these segments of conserved synteny are often preceded by conserved intergenic regions. Phylogenetic analysis of protein families shows several instances in which relatedness in the chemical structure of the glycopeptides is not reflected in the extent of the relationship of the corresponding polypeptides. Coherent branchings are observed for all polypeptides encoded by the syntenous gene cassettes. These results suggest that the acquisition of distinct, functional genetic elements has played a significant role in the evolution of glycopeptide gene clusters, giving them a mosaic structure. In addition, the synthesis of the structurally similar compounds A40926 and teicoplanin appears as the result of convergent evolution.

Total synthesis of the ristocetin aglycon.

The first total synthesis of the ristocetin aglycon is described employing a modular and highly convergent strategy. An effective 12-step (12% overall) synthesis of the ABCD ring system 3 from its amino acid subunits sequentially features an intramolecular aromatic nucleophilic substitution reaction for formation of the diaryl ether and closure of the 16-membered CD ring system (65%), a respectively diastereoselective (3:1, 86%) Suzuki coupling for installation of the AB biaryl linkage on which the atropisomer stereochemistry can be further thermally adjusted, and an effective macrolactamization (51%) for closure of the 12-membered AB ring system. A similarly effective 13-step (14% overall) synthesis of the 14-membered EFG ring system 4 was implemented employing a room-temperature intermolecular S(N)Ar reaction of an o-fluoronitroaromatic for formation of the FG diaryl ether (69%) and a key macrolactamization (92%) with formation of the amide linking residues 1 and 2. The two key fragments 3 and 4 were coupled, and the remaining 16-membered DE ring system was closed via diaryl ether formation to provide the ristocetin tetracyclic ring system (15 steps, 8% overall) enlisting an unusually facile (25 degrees C, 8 h, DMF, >/=95%) and diastereoselective (>/=15:1) aromatic nucleophilic substitution reaction that benefits from substrate preorganization.

Synthesis and evaluation of methyl ether derivatives of the vancomycin, teicoplanin, and ristocetin aglycon methyl esters.

A series of methyl ether derivatives of the vancomycin, teicoplanin, and ristocetin aglycon methyl esters was synthesized and their antimicrobial activity was established. These derivatives exhibit increased activity against VanB resistant strains of bacteria equipotent with that observed with sensitive bacteria.

Assembling the glycopeptide antibiotic scaffold: The biosynthesis of A47934 from Streptomyces toyocaensis NRRL15009.

The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the d-Ala-d-lactate ligase, vanAst, which resulted in the predicted -sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.

Inhibition of sporulation, glycopeptide antibiotic production and resistance in Streptomyces toyocaensis NRRL 15009 by protein kinase inhibitors.

Production of the glycopeptide antibiotic A47934 by Streptomyces toyocaensis NRRL 15009 begins in the late exponential phase in liquid culture and peaks in the early stationary phase. The pattern of cellular phosphoprotein production changes upon onset of A48934 production with the appearance of several novel phosphoproteins only when an antibiotic is being produced. Phosphoamino acid analysis revealed that S. toyocaensis NRRL 15009 produces proteins phosphorylated on His, Ser, Thr and Tyr, with most being membrane-associated. Addition of the isoflavones genistein or quercetin abolishes A47934 production in liquid culture and sporulation on solid medium. Furthermore, genistein slows the onset of inducible glycopeptide antibiotic resistance in S. toyocaensis NRRL 15009. These results support the participation of protein kinase pathways in A47934 biosynthesis and resistance and cell differentiation in S. toyocaensis NRRL 15009.

An analysis of the origins of a cooperative binding energy of dimerization.

The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

Trends in gram-positive bloodstream organism resistance: a seven-year audit of five glycopeptides and other drugs at a large university hospital.

Gram-positive pathogens (n = 525) isolated from bloodstream infections were tested by a reference broth microdilution method to establish antibiotic susceptibility trends (1985 to 1991). Cefazolin, ciprofloxacin, gentamicin, novobiocin, oxacillin, rifampin, teicoplanin, vancomycin, trimethoprim/sulfamethoxazole, daptomycin and two investigational glycopeptides (LY264826 and MDL62873) were investigated. Strains were selected without bias (first two isolates each month/species; no patient duplicates) as follows: 132 Staphylococcus aureus, 129 Staphylococcus epidermidis, 72 Staphylococcus haemolyticus, 130 Enterococcus faecalis and 62 other enterococci. All isolates were susceptible to vancomycin and teicoplanin except for Staphylococcus haemolyticus which had a resistant teicoplanin MIC90 of 64 micrograms/mL. The susceptibilities to vancomycin and teicoplanin were unchanged over the monitored period. Daptomycin, LY264826 and MDL62873 also demonstrated consistent activity; MDL62873 being superior with a MIC90 of 0.12 micrograms/mL. In 1990-1991 a significantly increased resistance to ciprofloxacin was observed among oxacillin-resistant strains of staphylococci. Our data suggest that the emergence of invasive vancomycin-resistant strains in Gram-positive isolates remains a rare phenomenon. However, we have experienced an emergence of numerous ciprofloxacin-resistant strains among staphylococci that precludes its empirical use at our institution.

Antistaphylococcal and antienterococcal activity of the new teicoplanin amide derivative MDL 62873.

In vitro response of 469 clinical isolates of gram-positive cocci was tested against MDL 62873 by the agar dilution method. The bacteria consisted of 407 isolates of staphylococci and 62 strains of enterococci. In vitro activity of MDL 62873 was compared with that of ampicillin, augmentin, erythromycin and vancomycin. All the isolates were completely inhibited by MDL 62873 at an MIC ranging between 0.25 and 8.0 micrograms/ml. In vitro activity of this new amide derivative of teicoplanin was far superior to that of ampicillin, augmentin and erythromycin and equal to or slightly better than that of vancomycin.

In vitro activity of mersacidin (M87-1551), an investigational peptide antibiotic tested against gram-positive bloodstream isolates.

We measured the in vitro activity of mersacidin (formerly M87-1551) against 183 clinical isolates (vancomycin susceptible) and 12 additional vancomycin-resistant strains of Gram-positive bacteria. The activity for mersacidin increased an average twofold (range, 1.7- to 7.6-fold) in a calcium-enriched medium. The minimum inhibitory concentration (MIC)90 for mersacidin was 8-32 times higher than vancomycin for staphylococci, 4-64 times higher for enterococci, and up to 32 times higher for other organisms tested. The MIC90 for MDL 62873, a comparison compound, was less than or equal to 0.5 micrograms/ml for all species except Staphylococcus haemolyticus (MIC90, 4 micrograms/ml), and it was greater than or equal to 4-fold more active than vancomycin. Against selected vancomycin-resistant strains, mersacidin had MICs greater than or equal to 16 micrograms/ml for enterococci, 4-32 micrograms/ml for Pediococcus, and less than or equal to 2 micrograms/ml for Leuconostoc species. Mersacidin may have some clinical utility in documented infections caused by staphylococci, nonenteric streptococci, Pediococcus, and Leuconostoc.

In vitro activities of three semisynthetic amide derivatives of teicoplanin, MDL 62208, MDL 62211, and MDL 62873.

MDL 62208, MDL 62211, and MDL 62873 are three semisynthetic amide derivatives of teicoplanin (MDL 62208 is an amide of teicoplanin aglycone, MDL 62211 is an amide of the teicoplanin A2 complex, and MDL 62873 is the corresponding derivative of peak A2-2 of the complex). The three semisynthetic glycopeptides were evaluated for in vitro antibacterial activity in comparison with the parent drug (teicoplanin) and vancomycin. A variety of gram-positive bacteria of clinical origin, whose species were carefully determined and that included 428 staphylococci (207 methicillin susceptible and 221 methicillin resistant), 41 streptococci, 82 enterococci, 43 strains of Listeria monocytogenes, 10 JK coryneform bacteria, and 67 anaerobes belonging to the genera Clostridium, Propionibacterium, Peptostreptococcus, and Eubacterium, were tested. The only resistances to MDL 62208, MDL 62211, and MDL 62873 were encountered with vancomycin- and teicoplanin-resistant enterococci. All of the other test strains, including some teicoplanin-resistant coagulase-negative staphylococci of the species Staphylococcus haemolyticus and Staphylococcus epidermidis, were highly susceptible to the three teicoplanin amides. Only minor differences in activity were observed among MDL 62208, MDL 62211, and MDL 62873, whereas the three experimental compounds were usually found to be more potent than teicoplanin or vancomycin (especially against staphylococci, with differences mostly ranging from 2- to 16-fold). The MBC-to-MIC ratios varied depending on the organisms, with the highest ratios usually observed for enterococci and listeriae. Overall, the MBC-to-MIC ratios yielded by the teicoplanin analogs were slightly greater than those yielded by teicoplanin or vancomycin.

Antimicrobial activity of MDL 62,873, a semisynthetic derivative of teicoplanin, in vitro and in experimental infections.

MDL 62,873 is an amide derivative of teicoplanin A2-2. Like those of natural glycopeptides, its antibacterial activity is mediated by inhibition of cell wall peptidoglycan synthesis. Against streptococci and enterococci, the in vitro activity of MDL 62,873 was similar to that of teicoplanin and greater than that of vancomycin. Against staphylococci, it has activity similar to that of vancomycin, and it was significantly more active than teicoplanin against coagulase-negative isolates. Like teicoplanin and vancomycin, MDL 62,873 had slow but significant bactericidal activity (99 to 99.9% killing in 24 h) against staphylococci at concentrations near the MIC. In murine septicemia studies with Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, the 50% effective doses were lower than those of vancomycin. In staphylococcal endocarditis in rats, MDL 62,873 at 20 mg/kg of body weight and vancomycin at 40 mg/kg, both doses given intravenously twice daily, had similar efficacies in reducing the heart bacterial load. These results probably reflect the longer half-life of MDL 62,873, which has a pharmacokinetic profile in rats similar to that of teicoplanin.

MM 55266 and MM 55268, glycopeptide antibiotics produced by a new strain of Amycolatopsis. Isolation, purification and structure determination.

Two novel glycopeptide antibiotics MM 55266 and MM 55268 containing fatty acid acyl functions, and of molecular formula C86H89N8O35Cl5 and C87H91N8O35Cl5, respectively, have been isolated and identified from a complex produced by Amycolatopsis sp. NCIB 40089. Fermentation conditions for their production, and methods for their isolation are described. Structures have been deduced by use of COSY and NOE NMR techniques and supported by chemical degradation studies. Both glycopeptides possessed good antibacterial activity against Gram-positive organisms.

In-vitro effects of teicoplanin, teicoplanin derivative MDL 62211 and vancomycin on human polymorphonuclear cell function.

The in-vitro effects on human neutrophil (PMN) functions of three structurally related glycopeptide antibiotics, vancomycin, teicoplanin and the teicoplanin derivative MDL 62211 were investigated. Teicoplanin and MDL 62211 significantly inhibited adherence, chemotaxis, phagocytosis and killing of Candida albicans by PMN's at a concentration of 500 mg/l, whereas PMN viability was only affected at drug concentrations of 2000 mg/l. Vancomycin interfered with PMN adherence and phagocytosis only at a concentration of 2000 mg/l without affecting PMN viability. Chemotaxis and killing of C. albicans were also not affected by this concentration. Teicoplanin and the teicoplanin-derivative MDL 62211 was found to have adverse effects on selected indices of PMN function in vitro only at concentrations higher than those employed in therapy, while vancomycin interfered only at very high concentrations.

Activities of two new teicoplanin amide derivatives (MDL 62211 and MDL 62873) compared with activities of teicoplanin and vancomycin against 800 recent staphylococcal isolates from France and the United States.

MDL 62211 is the amide derivative of the teicoplanin complex and MDL 62873 is a more focused amide derivative of the teicoplanin A2-2 peak. Each investigational compound had nearly identical activity and was 2- to 16-fold more active than teicoplanin or vancomycin. The MDL 62873 MICs for 90% of the strains tested were as follows: Staphylococcus aureus, oxacillin susceptible, 0.12 micrograms/ml; S. aureus, oxacillin resistant, 0.25 micrograms/ml; coagulase-negative staphylococci (CNS), oxacillin susceptible, 0.25 micrograms/ml; and CNS, oxacillin resistant, 2 micrograms/ml. CNS isolates from France were generally more susceptible than those tested in the United States. Teicoplanin-resistant U.S. isolates were usually Staphylococcus haemolyticus (1.8% of all tested strains), for which MICs ranged from 32 to greater than 128 micrograms/ml. MDL 62873 was not active against the Bacteroides fragilis group but was generally effective against gram-positive anaerobic strains.