Anatoly Severin

Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.

A pneumococcal clinical isolate with high-level resistance to cefotaxime and ceftriaxone.

A beta-lactam-resistant serotype 23F Streptococcus pneumoniae clinical isolate from the cerebrospinal fluid of a pediatric patient from California is unusual in that the MICs of cefotaxime and ceftriaxone (2.5 micrograms/ml each) are higher than that of benzylpenicillin (0.3 micrograms/ml); the isolate also has patterns of penicillin-binding proteins and of cell wall peptides which are atypical compared with those of previously examined penicillin-resistant pneumococci. Images

Mechanism of Action of the Mannopeptimycins, a Novel Class of Glycopeptide Antibiotics Active against Vancomycin-Resistant Gram-Positive Bacteria

ABSTRACT The naturally occurring mannopeptimycins (formerly AC98-1 through AC98-5) are a novel class of glycopeptide antibiotics that are active against a wide variety of gram-positive bacteria. The structures of the mannopeptimycins suggested that they might act by targeting cell wall biosynthesis, similar to other known glycopeptide antibiotics; but the fact that the mannopeptimycins retain activity against vancomycin-resistant organisms suggested that they might have a unique mode of action. By using a radioactive mannopeptimycin derivative bearing a photoactivation ligand, it was shown that mannopeptimycins interact with the membrane-bound cell wall precursor lipid II [C55-MurNAc-(peptide)-GlcNAc] and that this interaction is different from the binding of other lipid II-binding antibiotics such as vancomycin and mersacidin. The antimicrobial activities of several mannopeptimycin derivatives correlated with their affinities toward lipid II, suggesting that the inhibition of cell wall biosynthesis was primarily through lipid II binding. In addition, it was shown that mannopeptimycins bind to lipoteichoic acid in a rather nonspecific interaction, which might facilitate the accumulation of antibiotic on the bacterial cell surface.

Further Evidence that a Cell Wall Precursor [C55-MurNAc-(Peptide)-GlcNAc] Serves as an Acceptor in a Sorting Reaction

Previous studies suggested that a Gly-containing branch of cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus.

A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.

3,5-Dioxopyrazolidines, Novel Inhibitors of UDP-N- Acetylenolpyruvylglucosamine Reductase (MurB) with Activity against Gram-Positive Bacteria

ABSTRACT A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 μM, 4.3 to 10.3 μM, and 6.8 to 29.4 μM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 μM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 Å resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 μM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 μg/ml) and 4 (MICs, 4 to 8 μg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

2-Phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives as new inhibitors of bacterial cell wall biosynthesis

Twenty-five 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives were synthesized for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good inhibitory activity against Staphylococcus aureus MurB, MurC and MurD enzymes in vitro and antimicrobial activity against gram-positive bacteria including MRSA, VRE and PRSP. However, when they were tested in the presence of 4% bovine serum albumin, the MIC values increased to greater than 128 microg/mL against PRSP. None of the compounds demonstrated activity against gram-negative bacteria at MIC <32 microg/mL.

Penicillin-binding protein 2 is essential for expression of high-level vancomycin resistance and cell wall synthesis in vancomycin-resistant Staphylococcus aureus carrying the enterococcal vanA gene complex.

ABSTRACT A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in Staphylococcus aureus. The transcription of pbp2 of the highly vancomycin- and oxacillin-resistant strain COLVA200 and its mutant derivative with inactivated mecA were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-β-d-thiogalactopyranoside inducer was determined. The results indicate that mecA—the genetic determinant of oxacillin resistance—while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of mecA, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant S. aureus (A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem. 279:3398-3407, 2004) is penicillin binding protein 2.

Naphthyl tetronic acids as multi-target inhibitors of bacterial peptidoglycan biosynthesis.

Since the discovery of penicillin in 1929, many important antibiotic agents have made significant contributions to the prevention and treatment of infections caused by bacteria. Despite these remarkable achievements, infections are still the second-leading cause of death worldwide and remain a major public health problem. Clearly, there is great need for novel antibacterial agents to address resistance problems associated with current antibiotics. Toward this end, three broad strategies have been recently employed in the search for new leads: high-throughput screening of large compound libraries, genomics, and combinatorial biosynthesis. Although some limitations of the former approach to targets in bacterial peptidoglycan biosynthesis have been reported, the peptidoglycan biosynthetic pathway remains an attractive target, validated in the clinic with fosfomycin and vancomycin. Peptidoglycan biosynthesis is a complex process, which involves three main stages: a) cytoplasmic soluble enzymes that include MurA–F, b) membrane-bound enzymes that include MraY and MurG, and finally c) transglycosylases and transpeptidases, which act external to the cytoplasmic membrane. The Mur enzymes are unique to bacteria and are involved in essential functions of both Gram-positive and Gram-negative organisms. Another attractive aspect of Mur enzyme inhibitors is the potential to be bactericidal, leading to cell lysis and bacterial death. Inhibitors of peptidoglycan biosynthesis initiate a complex process of gene expression resulting in the induction of MurA and Mur I in Gram-positive bacteria to compensate for the slower rate of peptidoglycan biosynthesis. Several classes of natural products or their semisynthetic derivatives represented by liposidomycins, amphomycins, and muraymycins are inhibitors of MraY, whereas nisin, ramoplanin, and mersacidin are lipid II inhibitors. In the last decade a few small-molecule inhibitors of the Mur enzymes have been reported, including sesquiterpene lactones, 5-sulfonoxyanthranilic acids T6361 and T6362, UDP-MurNAc (MurA), imidazolinones, 4-thiazolidinones, thienopyrazoles, phosphinates (MurB), peptidosulfonamides, 3-cyanothiophenes (MurF), and d-glutamic acid analogues (Mur I). Despite the discovery of small-molecule inhibitors of various Mur enzymes, many limitations have been noted, including poor antibacterial activities in cells. In parallel, a number of new assay formats for the identification of Mur enzyme inhibitors have been described based on different platforms such as ultra-efficient affinity HTS, LC– MS, TLC, HPLC, and solid-support TLC. Our efforts in identifying Mur enzyme inhibitors were based on an initial pathway screen searching for inhibitors of multiple enzymes, MurA–F. Hits in this assay were evaluated against the individual Mur enzymes for lead optimization. Using this strategy, we identified two classes of inhibitors : 3,5-dioxopyrazolidines and pulvinones, with activities against several of the Mur enzymes. Inhibitors of multiple Mur enzymes are attractive given the essential role of each Mur enzyme in peptidoglycan biosynthesis. This strategy may prevent the development of drug resistance through the multi-target hypothesis. Herein we report on the SAR of the naphthyl tetronic acids and highlight their binding to the E. coli enzyme MurB. The target naphthylfuran-2-ones 5a–k were prepared by a three-step process starting from 3-bromo-4-methoxy-5H-furan2-one (1) and the appropriately substituted aldehydes 2 (Scheme 1). Bromofuranone 1 was acquired by bromination of the commercially available 4-methoxy-5H-furan-2-one with Nbromosuccinimide in carbon tetrachloride at reflux. Deprotonation of 2 at C5 with lithium isopropylcyclohexylamide (LICA) followed by an aldol reaction with substituted aldehydes 2 mediated by anhydrous ZnCl2 afforded diastereomeric alcohols, which were converted into their mesylate or chloride derivatives in situ followed by elimination to generate the exocyclic double bond of 3 in the thermodynamically more stable Z configuration. The key step involved a Suzuki cross-coupling of 3 with aryl boronic acids catalyzed by either [PdACHTUNGTRENNUNG(PPh3)4] or [PdCl2ACHTUNGTRENNUNG(dppf)2] to afford the methoxyfuranones 4a–k. Demethylation of methoxyfuranones 4a–k with lithium bromide in the final step afforded the desired naphthylfuran-2ones 5a–k. Purification by silica gel column chromatography was followed by an acid wash of the collected fractions to restore the acidic functionality. A panel of nine enzymes was used to assess the abilities of the naphthylfuranones to inhibit the Mur enzymes, and thus to define SAR trends for multiple enzyme inhibition (Table 1). The four isomeric bis-naphthyl compounds (Entries 1–4) were evaluated to determine whether there is a preference for aor blinked naphthyl groups at either C3 or C5. The trend seems to favor C3 b and C5 a substitution. Replacement of naphthyl with p-chlorophenyl (Entries 5–8) gave compounds 5e–h with good broad-spectrum activity against the Mur enzymes, thus confirming the desired SAR trend. Further optimization of the C5 a methylidene naphthyl derivative 5h by changing the p[a] Dr. T. S. Mansour, Dr. B. Rasmussen, Dr. G. Krishnamurthy, C. Smeltzer, Dr. Y. Yang, Dr. A. Severin, P. J. Petersen, Dr. G. Singh Medicinal Chemistry, Wyeth Research 401 North Middletown Road, Pearl River, NY 10965 (USA) Fax: (+1)845-602-5580 E-mail : mansout@wyeth.com [b] Dr. C. E. Caufield, K. M. Morris, S. Naughton, S. Antane, Dr. D. Quagliato Wyeth Research, CN 8000, Princeton, NJ 08543 (USA) [c] Dr. R. Chopra, K. Svenson, Dr. J. Bard Wyeth Research, Cambridge, MA 02140 (USA)

The Structure of the Cell Wall Peptidoglycan of Bacillus cereus RSVF1, a Strain Closely Related to Bacillus anthracis

The peptidoglycan of Bacillus cereus RSVF1, a close relative of Bacillus anthracis, has several distinguishing features: the overwhelming majority of cross-linked muropeptides are dimers, higher oligomers are only present in minute quantities; and virtually all muropeptides lack the N-acetyl group from glucosamine residues, thus explaining resistance of the cell walls to lysozyme.