Search

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin., (© 2013 The Authors. Published by Wiley‐VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of Creative Commons the Attribution Non‐Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.)

To make progress in understanding peptidoglycan metabolism, we will reconstitute in vitro the assembly process and the molecular machineries that carry out this formidable task. We review here the reports of isolation of complexes comprising penicillin-binding proteins (PBPs), the enzymes that synthesize the peptidoglycan from its lipid-linked precursor.

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.

The mechanisms by which bacteria adopt and maintain individual shapes remain enigmatic. Outstanding questions include why cells are a certain size, length, and width; why they are uniform or irregular; and why some branch while others do not. Previously, we showed that Escherichia coli mutants lacking multiple penicillin binding proteins (PBPs) display extensive morphological diversity. Because defective sites in these cells exhibit the structural and functional characteristics of improperly localized poles, we investigated the connection between cell division and shape. Here we show that under semipermissive conditions the temperature-sensitive FtsZ84 protein produces branched and aberrant cells at a high frequency in mutants lacking PBP 5, and this phenotype is exacerbated by the loss of additional peptidoglycan endopeptidases. Surprisingly, certain ftsZ84 strains lyse at the nonpermissive temperature instead of filamenting, and inhibition of wild-type FtsZ forces some mutants into tightly wound spirillum-like morphologies. The results demonstrate that significant aspects of bacterial shape are dictated by a previously unrecognized relationship between the septation machinery and ostensibly minor peptidoglycan-modifying enzymes and that under certain circumstances improper FtsZ function can destroy the structural integrity of the cell.

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.

The enzymes involved in the biosynthesis of peptidoglycan are targets for the development of new antibiotics. The bifunctional high molecular weight (HMW) penicillin-binding proteins (PBPs), which contain both glycosyltransferase (GTase) and transpeptidase (TPase) activities, are particularly attractive targets because of their extracellular location. However, there is limited mechanistic or structural information about the GTase modules of these enzymes. In this paper, we describe the overexpression and characterization of the GTase module of Escherichia coli PBP1b, a paradigm of the HMW PBPs. We define the C-terminal boundary of the GTase module and show that the isolated module can be overexpressed at significantly higher levels than the full-length protein. The catalytic efficiency and other characteristics of the isolated module are comparable in most respects to the full-length enzyme. This work lays the groundwork for mechanistic and structural analysis of GTase modules.

The SOS response aids bacterial propagation by inhibiting cell division during repair of DNA damage. We report that inactivation of the ftsI gene product, penicillin binding protein 3, by either beta-lactam antibiotics or genetic mutation induces SOS in Escherichia coli through the DpiBA two-component signal transduction system. This event, which requires the SOS-promoting recA and lexA genes as well as dpiA, transiently halts bacterial cell division, enabling survival to otherwise lethal antibiotic exposure. Our findings reveal defective cell wall synthesis as an unexpected initiator of the bacterial SOS response, indicate that beta-lactam antibiotics are extracellular stimuli of this response, and demonstrate a novel mechanism for mitigation of antimicrobial lethality.

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

ME1036, formerly CP5609, is a novel parenteral carbapenem with a 7-acylated imidazo[5,1-b]thiazole-2-yl group directly attached to the carbapenem moiety of the C-2 position. The present study evaluated the in vitro activities of ME1036 against clinical isolates of gram-positive and gram-negative bacteria. ME1036 displayed broad activity against aerobic gram-positive and gram-negative bacteria. Unlike other marketed beta-lactam antibiotics, ME1036 maintained excellent activity against multiple-drug-resistant gram-positive bacteria, such as methicillin-resistant staphylococci and penicillin-resistant Streptococcus pneumoniae (PRSP). The MICs of this compound at which 90% of isolates were inhibited were 2 microg/ml for methicillin-resistant Staphylococcus aureus (MRSA), 2 microg/ml for methicillin-resistant coagulase-negative staphylococci, and 0.031 microg/ml for PRSP. In time-kill studies with six strains of MRSA, ME1036 at four times the MIC caused a time-dependent decrease in the numbers of viable MRSA cells. The activity of ME1036 against MRSA is related to its high affinity for penicillin-binding protein 2a, for which the 50% inhibitory concentration of ME1036 was approximately 300-fold lower than that of imipenem. In conclusion, ME1036 demonstrated a broad antibacterial spectrum and high levels of activity in vitro against staphylococci, including beta-lactam-resistant strains.

In vitro activities of ABT-492, ciprofloxacin, levofloxacin, trovafloxacin, moxifloxacin, gatifloxacin, and gemifloxacin were compared. ABT-492 was more potent against quinolone-susceptible and -resistant gram-positive organisms, had activity similar to that of ciprofloxacin against certain members of the family Enterobacteriaceae, and had comparable activity against quinolone-susceptible, nonfermentative, gram-negative organisms. Bactericidal activity of ABT-492 was also evaluated.

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.

Extensive use of beta-lactam antibiotics has led to the selection of pathogenic streptococci resistant to beta-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 beta-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S(443)SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.

The in vitro activities of piperacillin (PIP) against beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae were compared with those of cefotaxime (CTX) and ceftriaxone (CRO), and the potency of PIP as therapy for meningitis caused by BLNAR is also discussed. PIP showed good activity (MIC at which 90% of strains are inhibited, 0.25 micro g/ml) against 69 BLNAR strains, and its activity was comparable to that of CRO and superior to that of CTX. No significant correlation was observed between the MICs of PIP and CTX or CRO or AMP, whereas a high correlation was observed between the MICs of CTX and CRO. In the killing study, PIP showed potent bactericidal activity compared with those of CTX and CRO. By microscopic examination, PIP caused the formation of a spindle and short filamentous cells with bulges and induced cell lysis in BLNAR strains, while treatment with CTX and CRO resulted in the formation of large, spherical cells without any obvious lysis. The affinity of Bocillin FL, a fluorescent penicillin used for determination of the 50% inhibitory concentration (IC(50)s) for penicillin-binding proteins (PBPs), to PBPs 3a and 3b of BLNAR strains was drastically decreased compared with that to an AMP-susceptible strain (ATCC 33391). In the case of the BLNAR strains, the IC(50)s for PBPs 1a, 1b, and 2 were similar to those for the PBPs of ATCC 33391. Since the affinity of binding to PBPs 3a and 3b of the BLNAR strains decreased drastically, the second targets among the PBPs were PBP 2 for PIP, PBP1 (1a and 1b) for CTX and CRO. In conclusion, PIP showed excellent activities against BLNAR strains in a manner different from those of cephem antibiotics, suggesting that it could be a candidate therapeutic agent for the treatment of meningitis caused by BLNAR strains.

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.

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 [C(55)-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.

The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

The major constituent of the bacterial cell wall, peptidoglycan, is comprised of repeating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) with an appended peptide. Penicillin-binding proteins (PBPs) are involved in the final stages of bacterial cell wall assembly. Two activities for PBPs are the cross-linking of the cell wall, carried out by dd-transpeptidases, and the dd-peptidase activity, that removes the terminal d-Ala residue from peptidoglycan. The dd-peptidase activity moderates the extent of the cell wall cross-linking. There exists a balance between the two activities that is critical for the well-being of bacterial cells. We have cloned and purified PBP5 of Escherichia coli. The membrane anchor of this protein was removed, and the enzyme was obtained as a soluble protein. Two fragments of the polymeric cell wall of Gram-negative bacteria (compounds 5 and 6) were synthesized. These molecules served as substrates for PBP5. The products of the reactions of PBP5 and compounds 5 and 6 were isolated and were shown to be d-Ala and the fragments of the substrates minus the terminal d-Ala. The kinetic parameters for these enzymic reactions were evaluated. PBP5 would appear to have the potential for turnover of as many as 1.4 million peptidoglycan strands within a single doubling time (i.e., generation) of E. coli.

Bacterial cell shape is determined by a rigid external cell wall. In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP-PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.

Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit beta-lactamases to release triclosan through hydrolysis of the beta-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A beta-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C beta-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k(cat)/K(m)). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the beta-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the beta-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for beta-lactam binding, DD-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30,000 m-1.s-1). Against a set of five alpha- and epsilon-substituted l-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N epsilon-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N epsilon-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac2-l-Lys-D-Ala-d-Ala (kcat/Km) was bell-shaped, with pKa values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs.

Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus(MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2 '(2a) (PBP2 '(PBP2a)) which mainly confers the resistance to beta-lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2 '(PBP2a) slightly. Using BOCILLIN FL, a fluorescent-labeled benzyl penicillin, we found that PBP2 '(PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2 '(PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of beta-lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of beta-lactamase to some extent.

The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing sigma(E). Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.

Penicillin-binding proteins (PBPs) are responsible for the final stages of bacterial cell wall assembly. These enzymes are targets of beta-lactam antibiotics. Two of the PBP activities include dd-transpeptidase and DD-carboxypeptidase activities, which carry out the cross-linking of the cell wall and trimming of the peptidoglycan, the major constituent of the cell wall, by an amino acid, respectively. The activity of the latter enzyme moderates the degree of cross-linking of the cell wall, which is carried out by the former. Both these enzymes go through an acyl-enzyme species in the course of their catalytic events. Compound 6, a cephalosporin derivative incorporated with structural features of the peptidoglycan was conceived as an inhibitor specific for DD-transpeptidases. On acylation of the active sites of dd-transpeptidases, the molecule would organize itself in the two active site subsites such that it mimics the two sequestered strands of the bacterial peptidoglycan en route to their cross-linking. Hence, compound 6 is the first inhibitor conceived and designed specifically for inhibition of DD-transpeptidases. The compound was synthesized in 13 steps and was tested with recombinant PBP1b and PBP5 of Escherichia coli, a dd-transpeptidase and a dd-carboxypeptidase, respectively. Compound 6 was a time-dependent and irreversible inhibitor of PBP1b. On the other hand, compound 6 did not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate.

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.

The purpose of this study was to examine the percentage of Beta-lactam-resistant streptococcal carriers in healthy adults, and to investigate the relationships among minimum inhibitory concentrations (MICs) of Beta-lactams, alterations in the penicillin-binding protein genes ( pbp genes), and the affinity of penicillin-binding proteins (PBPs) for ampicillin (ABPC) in Streptococcus mitis. We also compared numbers of surviving bacteria at various ABPC concentrations in both ABPC-susceptible and -resistant S. mitis strains. The percentages of subjects carrying ABPC- and cefaclor (CCL)-resistant streptococci were 52% (27 of 52 subjects) and 100%, respectively. S. mitis, including both antibiotic-susceptible and -resistant strains, were classified into five groups according to the pbp gene mutations that resulted in alterations of the deduced amino-acid sequence in the homology boxes of PBPs. All ABPC-resistant strains showed alterations in PBP1A, 2X, and 2B, while no or only PBP2X alterations were detected in the susceptible strains. These results suggest that the accumulation of pbp gene mutations is strongly related to the MIC of ABPC for S. mitis. In the resistant strains, the affinity of PBPs for ABPC was reduced in comparison with that in the susceptible strains, and the bactericidal effect of ABPC was also reduced. Therefore, we should be aware of conditions such as infective endocarditis that are caused by Beta-lactam-nonsusceptible streptococci in the normal oral flora.

The roles of beta-lactamase and alterations in penicillin-binding protein in the development of amoxicillin and amoxicillin/clavulanate resistance in two beta-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains of Haemophilus influenzae were investigated. Seven beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were also studied for comparison of their resistance mechanisms. All strains had been recovered from patients in Japan. The TEM type beta-lactamase of the two BLPACR strains had 100% homology with the amino acid sequences of published TEM-1 beta-lactamase, showing that amoxicillin/clavulanate resistance was not associated with mutations in this beta-lactamase. However, these strains, as well as the seven BLNAR strains, had multiple mutations in ftsI, which encodes penicillin binding protein 3 (PBP3). The transformation of H. influenzae Rd strain with amplified ftsI genes from two BLPACR and two BLNAR strains enabled the selection of amoxicillin/clavulanate-resistant transformants with the same mutations as their parent strains. We concluded that amoxicillin/clavulanate resistance in the two BLPACR strains was due to changes in PBP3. The possibility of the presence of an extended spectrum beta-lactamase was excluded in the BLPACR strains studied.

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.

We have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram-positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell division sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell division protein FtsZ, which recruits penicillin-binding protein (PBP) 2. Unexpectedly, in FtsZ-depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell division proteins as drug targets.

beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate expression of these inducible proteins. Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence to the medically significant Staphyloccocal BlaI and MecI proteins. This structure is composed of a compact core of three alpha-helices and a three-stranded beta-sheet typical of the winged helix protein (WHP) family. The protein/DNA complex was studied by NMR chemical shift comparison between the free and complexed forms of BlaI. Residues involved in DNA interaction were identified and a WHP canonical model of interaction with the operators is proposed. In this model, specific contacts occur between the base-pairs of the TACA motif and conserved amino acid residues of the repressor helix H3. These results help toward understanding the repression and induction mechanism of the genes coding for beta-lactamase and PBP2'.

Amphiphilic alpha-helices play a fundamental role in protein membrane association and show a segregation of polar and apolar amino acid residues. Based on correlations between amphiphilic properties and biological function, a number of theoretical approaches have been developed, which quantify alpha-helix amphiphilicity and then attempt to assign function. The most commonly used measure of amphiphilicity is the hydrophobic moment, < microH >, which, when used in conjunction with an alpha-helix's mean hydrophobicity, < H >, has been used to classify membrane interactive amphiphilic alpha-helices as either surface active or transmembrane. Here, the predictive efficacy of plot methodology is reviewed by examining published data, which compare the function of known membrane interactive amphiphilic alpha-helices to that assigned by this methodology. The results of this review are discussed in relation to the reliability of < microH > as a quantifier of alpha-helical amphiphilicity, and the ability of < microH > and < H > to describe alpha-helical structure / function relationships. It is concluded that hydrophobic moment plot methodology is not a generally reliable predictor of alpha-helical structure / function relationships. It appears that the inefficacy of plot methodology is primarily due to the inability of the plot diagram to accommodate the heterogeneity of the alpha-helical classes it attempts to define. However, the predictive efficacy of the methodology appears to be improved if other alpha-helical parameters are also considered when assigning alpha-helical function. It is suggested that the conventional methodology should be seen only as an indicator for the assignation of structure / function relationships, providing a guide to future experimental investigations.

The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the beta-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of E. hirae to beta-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in beta-lactam resistance or in the regulation of cell autolysis in E. hirae.

Beta-lactam antibiotics inhibit enzymes involved in the last step of peptidoglycan synthesis. These enzymes, also identified as penicillin-binding proteins (PBPs), form a long-lived acyl-enzyme complex with beta-lactams. Antibiotic resistance is mainly due to the production of beta-lactamases, which are enzymes that hydrolyze the antibiotics and so prevent them reaching and inactivating their targets, and to mutations of the PBPs that decrease their affinity for the antibiotics. In this study, we present a theoretical study of several penicillin-recognizing proteins complexed with various beta-lactam antibiotics. Hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations have been performed to understand the role of several residues, and pK(a) calculations have also been done to determine their protonation state. We analyze the differences between the beta-lactamase TEM-1, the membrane-bound PBP2x of Streptococcus pneumoniae, and the soluble DD-transpeptidase of Streptomyces K15., (Copyright 2003 Wiley-Liss, Inc.)

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and beta-lactamases, resistance enzymes to beta-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for beta-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7beta-[N-Acetyl-L-alanyl-gamma-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC beta-lactamase from Escherichia coli was solved at 1.71 A resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the beta-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the beta-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.

The bacterial peptidoglycan consists of glycan chains of repeating beta-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The beta-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K(m) and k(cat)/K(m) were measured at 40.6 micro M (+/- 15.5) and 1 x 10(-3) M(-1) s(-1), respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

Streptomyces cacaoi beta-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb beta-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of beta-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.

Objectives: To investigate high-level piperacillin resistance in Veillonella spp. in the absence of beta-lactamase activity., Methods: Penicillin-binding protein (PBP) competition studies were conducted in Veillonella strains, with piperacillin MICs ranging from 0.5 to >128 mg/L and ampicillin MICs from 0.125 to 4 mg/L. Whole cell lysates were pre-incubated with piperacillin or ampicillin and post-labelled with [3H]benzylpenicillin., Results: PBP competition studies showed that the PBP with greatest affinity for penicillin and ampicillin had a molecular weight of approximately 66 kDa, and exhibited reduced binding of piperacillin in resistant strains., Conclusions: This unusual focusing of different penicillins on one PBP may be the cause of selective mutants resulting from piperacillin MICs > 128 mg/L. In the absence of beta-lactamases, alterations in penicillin-binding were seen to be major contributors to high-level piperacillin resistance development.

The transcription of mecA, the gene required for oxacillin resistance in staphylococci, was quantified in a collection of 65 geographically and genetically diverse clinical and 8 defined laboratory Staphylococcus aureus isolates. mecA transcription was measured by real-time reverse transcription-PCR, confirmed by Northern blot analysis, and correlated with the presence and DNA sequence of the two mecA repressors, mecI and blaI. Isolates were first examined that contained mecI and/or blaI with wild-type sequence. BlaI provided significantly more repression of mecA transcription than did MecI, unrelated to blaI genetic location. Both together repressed mecA better than either one alone. In clinical isolates containing only wild-type mecI, mecA transcription repression was 10- to 25-fold less effective than that seen in previously studied constructs derived from strain N315. There was a difference in the mecI ribosomal binding site (RBS) between the clinical isolates (GGAA) and N315 (GGAG). The GGAA RBS was associated with 5.5- to 7.3-fold less mecA repression than GGAG in isogenic constructs. The values generated for wild-type repressors were compared to those in 26 isolates containing mecI mutations. mecA transcription appeared to be repressed only by BlaI in isolates with mecI nonsense and frameshift mutations. In contrast, mecI repression seemed to be partially or fully retained in many of the isolates with mecI and one isolate with blaI missense mutations, providing structure-function correlates with the site and type of mutation. We conclude that mecA repressor activity is highly variable in clinical S. aureus isolates due to mecI mutations, RBS polymorphisms, and unidentified genomic adaptations.

Crystalline 1 (TAK-599) is a novel N-phosphono prodrug of anti-methicillin-resistant Staphylococcus aureus (MRSA) cephalosporin 2a (T-91825) that has high affinity for penicillin-binding protein (PBP) 2' (IC(50); 0.90 microg/mL) and shows potent in vitro anti-MRSA activity (MIC against MRSA N133; 1.56 microg/mL), comparable to that of vancomycin (1.56 microg/mL). Although 2a had insufficient water solubility (2.3 mg/mL) for parenteral administration, 1 showed excellent water solubility (>100 mg/mL, pH 7) as well as good chemical stability in the solid state and solution. In pharmacokinetic studies, when 1 was administered intravenously to rats and monkeys, it was rapidly converted into 2a in the blood. These results show that 1 (TAK-599) is a highly promising parenteral cephalosporin targeted for MRSA infection.

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.

AM-112 [(1'R,5R,6R)-3-(4-amino-1,1-dimethyl-butyl)-6-(1'-hydroxyethyl)oxapenem-3-carboxylate] is a novel oxapenem compound which possesses potent beta-lactamase-inhibitory properties. Fifty-percent inhibitory concentrations (IC(50)s) of AM-112 for class A enzymes were between 0.16 and 2.24 micro M for three enzymes, compared to IC(50)s of 0.008 to 0.12 micro M for clavulanic acid. Against class C and class D enzymes, however, the activity of AM-112 was between 1,000- and 100,000-fold greater than that of clavulanic acid. AM-112 had affinity for the penicillin-binding proteins (PBPs) of Escherichia coli DC0, with PBP2 being inhibited by the lowest concentration of AM-112 tested, 0.1 micro g/ml. Ceftazidime was combined with AM-112 at 1:1 and 2:1 ratios in MIC determination studies against a panel of beta-lactamase-producing organisms. These studies demonstrated that AM-112 was effective at protecting ceftazidime against extended-spectrum beta-lactamase-producing strains and derepressed class C enzyme producers, reducing ceftazidime MICs by 16- and 2,048-fold. Similar results were obtained when AM-112 was combined with ceftriaxone, cefoperazone, or cefepime in a 1:2 ratio. Protection of ceftazidime with AM-112 was maintained against Enterobacter cloacae P99 and Klebsiella pneumoniae SHV-5 in a murine intraperitoneal sepsis model. The 50% effective dose of ceftazidime against E. cloacae P99 and K. pneumoniae SHV-5 was reduced from >100 and 160 mg/kg of body weight to 2 and 33.6 mg/kg, respectively, when it was combined with AM-112 at a 1:1 ratio. AM-112 demonstrates potential as a new beta-lactamase inhibitor.

Penicillin binding protein (PBP) 5, a DD-carboxypeptidase that removes the terminal D-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp(218) and Lys(219) residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.

The in vitro antibacterial activity of S-3578, a new parenteral cephalosporin, against clinical isolates was evaluated. The MICs of the drug at which 90% of the isolates were inhibited were 4 micro g/ml for methicillin-resistant Staphylococcus aureus (MRSA) and 2 micro g/ml for methicillin-resistant Staphylococcus epidermidis, which were fourfold higher than and equal to those of vancomycin, respectively. The anti-MRSA activity of S-3578 was considered to be due to its high affinity for penicillin-binding protein 2a (50% inhibitory concentration, 4.5 micro g/ml). In time-kill studies with 10 strains each of MRSA and methicillin-susceptible S. aureus, S-3578 caused more than a 4-log(10) decrease of viable cells on the average at twice the MIC after 24 h of exposure, indicating that it had potent bactericidal activity. Furthermore, in population analysis of MRSA strains with heterogeneous or homogeneous resistance to imipenem, no colonies emerged from about 10(9) cells on agar plates containing twice the MIC of S-3578, suggesting the low frequency of emergence of S-3578-resistant strains from MRSA. S-3578 was also highly active against penicillin-resistant Streptococcus pneumoniae (PRSP), with a MIC(90) of 1 micro g/ml, which was comparable to that of ceftriaxone. S-3578 also had antibacterial activity against a variety of gram-negative bacteria including Pseudomonas aeruginosa, though its activity was not superior to that of cefepime. In conclusion, S-3578 exhibited a broad antibacterial spectrum and, particularly, had excellent activity against gram-positive bacteria including methicillin-resistant staphylococci and PRSP. Thus, S-3578 was considered to be worthy of further evaluation.