Resistance to beta-lactam antibiotics and its mediation by the sensor domain of the transmembrane BlaR signaling pathway in Staphylococcus aureus

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.     

Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.     

Uptake of dipeptide and beta-lactam antibiotics by the basolateral membrane vesicles prepared from rat kidney

The transport of dipeptides and beta-lactam antibiotics across the rat renal basolateral membrane was examined. The initial uptake of glycylsarcosine and cefadroxil by rat renal basolateral membrane vesicles was inhibited by the presence of all the di- and tripeptides and beta-lactam antibiotics that were tested in this study. However, the uptake of both substrates was not inhibited by glycine, an amino acid. The initial uptake of zwitterionic beta-lactam antibiotics, cefadroxil, cephradine, and cephalexin, was stimulated by preloaded glycylsarcosine (countertransport effect). On the other hand, the uptake of dianionic beta-lactam antibiotics, ceftibuten and cefixime, was not affected. A concentration-dependent initial uptake of glycylsarcosine and cefadroxil suggested the existence of a carrier-mediated mechanism, whereas the transport of ceftibuten did not show any saturated uptake. The transporter that participates in the permeation of dipeptides and beta-lactam antibiotics across basolateral membranes showed lower affinity than did PEPT1 and PEPT2. This is the first study that showed an evidence for a peptide transporter, expressed in the rat renal basolateral membrane, that recognizes zwitterionic beta-lactam antibiotics using basolateral membrane vesicles isolated from normal rat kidney.     

Novel mechanism of hydrolysis of therapeutic beta-lactams by Stenotrophomonas maltophilia L1 metallo-beta-lactamase.

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.     

Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate.

Genetic and biochemical evidence was obtained for lysine catabolism via cadaverine and delta-aminovalerate in both the beta-lactam producer Streptomyces clavuligerus and the nonproducer Streptomyces lividans. This pathway is used when lysine is supplied as the sole source of nitrogen for the organism. A second pathway for lysine catabolism is present in S. clavuligerus but not in S. lividans. It leads to alpha-aminoadipate, a precursor for beta-lactam biosynthesis. Since it does not allow S. clavuligerus to grow on lysine as the sole nitrogen source, this pathway may be used exclusively to provide a precursor for beta-lactam biosynthesis. beta-Lactam producers were unable to grow well on alpha-aminoadipate as the only nitrogen source, whereas three of seven species not known to produce beta-lactam grew well under the same conditions. Lysine epsilon-aminotransferase, the initial enzyme in the alpha-aminoadipate pathway for lysine catabolism, was detected in cell extracts only from the beta-lactam producers. These results suggest that synthesis of alpha-aminoadipate is exclusively a secondary metabolic trait, present or expressed only in beta-lactam producers, while genes governing the catabolism of alpha-aminoadipate are present or fully expressed only in beta-lactam nonproducers.     

Metabolic engineering of beta-lactam production

Metabolic engineering has become a rational alternative to classical strain improvement in optimisation of beta-lactam production. In metabolic engineering directed genetic modification are introduced to improve the cellular properties of the production strains. This has resulted in substantial increases in the existing beta-lactam production processes. Furthermore, pathway extension, by heterologous expression of novel genes in well-characterised strains, has led to introduction of new fermentation processes that replace environmentally damaging chemical methods. This minireview discusses the recent developments in metabolic engineering and the applications of this approach for improving beta-lactam production.     

Effects of beta-lactam antibiotics on eukaryotic cells

A symposium on effects of beta-lactam antibiotics on eukaryotic cells was held as part of the 9th International Congress of Infectious and Parasitic Diseases (Munich, Germany, July 1986). This symposium provided an opportunity to review recent work on the effect of beta-lactam structures on mammalian cells in culture and to speculate on possible clinical implications. This paper is a comment on the subject matter covered by the symposium papers which follow.Abbreviations Ara-C cytosine arabinoside - BLA beta-lactam antibiotic     

Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine

The enzyme beta-lactam synthetase (beta-LS) catalyzes the formation of the beta-lactam ring in clavulanic acid, a clinically important beta-lactamase inhibitor. Whereas the penicillin beta-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, beta-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, beta-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a beta-LS. The 1.95 A resolution structure of Streptomyces clavuligerus beta-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in beta-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate beta-lactam formation. The structural data provide the opportunity to alter the synthetic potential of beta-LS, perhaps leading to the creation of new beta-lactamase inhibitors and beta-lactam antibiotics.     

Delta 2- and delta 3-cephalosporins, penicillinate and 6-unsubstituted penems. Intrinsic reactivity and interaction with beta-lactamases and D-alanyl-D-alanine-cleaving serine peptidases.

The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been expressed in terms of the second-order rate constant (M-1.s-1(OH-)) for the hydrolysis of the beta-lactam amide bond by OH- at 37 degrees C. The values thus obtained have been compared with the second-order rate constants (M-1.s-1(enzyme) for the opening of the same beta-lactam amide bond during interaction with the beta-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio M-1.s-1(enzyme)/M-1.s-1(OH) varies from less than 2 to more than 1 x 10(6). The primary parameter that governs enzyme action is the goodness of fit of the beta-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D-alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100 s, values significantly lower than those exhibited by most beta-lactam compounds.     

Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase

Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.     

Nature of monocyclic beta-lactam antibiotic Nocardicin A to beta-lactamases

Nocardicin A is the antibiotic which was first found to possess a monocyclic beta-lactam ring. This antibiotic was inactivated by the cleavage of its beta-lactam ring. The direct spectrophotometric assay was applied to measure the rate of enzymatic hydrolysis of Nocardicin A. Nocardicin A was highly stable to both chromosomal and plasmid-mediated beta-lactamases. Of the nine beta-lactam antibiotics including cefoxitin and cefuroxime, Nocardicin A was the most stable to the beta-lactamases tested excluding those from Klebsiella oxytoca and Proteus vulgaris. The latter broad-spectrum beta-lactamases hydrolyzed Nocardicin A rather intensively. Extreme stability of Nocardicin A to beta-lactamases was suggested to be due to the combination of its low affinity to the enzymes and stabilization of its monocyclic beta-lactam ring. Nocardicin A was shown to have inducing ability toward beta-lactamases.     

Development of DNA chip for the simultaneous detection of various beta-lactam antibiotic-resistant genes

A robust and fast DNA chip method was developed in order to detect the various beta-lactam antibiotic-resistance genes in one slide. These genes included PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY, and AmpC. beta-lactam antibiotic-resistance genes were labeled with a fluorescent nucleotide by a multiplex polymerase chain reaction using a mixture of specific primer sets for each gene. This labeled target was hybridized with a DNA chip that contained the spots of the specific probe DNAs for each beta-lactam antibiotic-resistance gene. This technique made it possible to detect the specific resistance gene, even in a single bacterium.     

Diversity of penicillin-binding proteins. Resistance factor FmtA of Staphylococcus aureus

Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all beta-lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by beta-lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with beta-lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and beta-lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for beta-lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to beta-lactam inactivation. We found that FmtA undergoes conformational changes in presence of beta-lactams that may be essential to the beta-lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under beta-lactam induced cell wall stress conditions.     

Second lytic target of beta-lactam compounds that have a terminal D-amino acid residue

A new biochemical mechanism of lysing bacterial cells by treatment with certain beta-lactam compounds that possess a terminal D-amino acid moiety in their side chain was demonstrated. The two functions of the molecule, the beta-lactam and terminal D-amino acid moiety, are both involved in the activity of lysing gram-negative bacteria, which is characterized by very rapid lysis of the cells in the first few hours after their contact with the compound. This mechanism was proved by studies on one such compound, named MT-141, which contains a terminal D-cysteine moiety with free amino and carboxyl groups in the 7 beta-side chain of the 7 alpha-methoxy-cephalosporin skeleton. This compound bound to the cell-wall peptidoglycan of Escherichia coli through the D-amino group of its terminal D-amino acid moiety and this seemed to cause rapid cell lysis. Both activities, of binding to peptidoglycan and of causing rapid cell lysis, were inhibited by certain D-amino acids, but not by L-amino acids. Mutants were isolated that had simultaneously gained decreased sensitivity to this kind of beta-lactam compound and supersensitivity to globomycin, an inhibitor of formation of lipoproteins which function in linking the peptidoglycan to the outer membrane. These results suggest that binding of the terminal D-amino acid moiety of the beta-lactam compound to peptidoglycan somehow influences formation of the linkage between the outer membrane and the peptidoglycan and consequently enhances the cell lytic activity of the beta-lactam portion of the molecule.     

beta-lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter

Therapeutic use of cephaloridine, a beta-lactam antibiotic, in humans is associated with carnitine deficiency. A potential mechanism for the development of carnitine deficiency is competition between cephaloridine and carnitine for the renal reabsorptive process. OCTN2 is an organic cation/carnitine transporter that is responsible for Na(+)-coupled transport of carnitine in the kidney and other tissues. We investigated the interaction of several beta-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The beta-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated carnitine transport. These antibiotics possess a quaternary nitrogen as does carnitine. Several other beta-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to carnitine. Interestingly, many of the beta-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H(+)-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na(+)-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na(+)-independent. Furthermore, the Na(+)-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain beta-lactam antibiotics such as cephaloridine and that cephaloridine-induced carnitine deficiency is likely to be due to inhibition of carnitine reabsorption in the kidney.     

Evolution of the clusters of genes for beta-lactam antibiotics: a model for evolutive combinatorial assembly of new beta-lactams.

beta-Lactam antibiotics are produced by prokaryotic and eukaryotic organisms. The genes for beta-lactam biosynthesis are organized in clusters but the location of the different genes is not identical. Biosynthesis genes are clustered with genes for resistance (bla, pbp) and for the efflux of the antibiotic (cmcT) in prokaryotes. Comparison of proteins reveals much larger differences for primary metabolism enzymes than for beta-lactam biosynthesis enzymes in producing organisms. This suggests a horizontal transfer of the beta-lactam antibiotic biosynthesis genes.     

Three-dimensional structures of enzymes useful for beta-lactam antibiotic production

Significant advances have been made in the structure-based engineering of enzymes useful for beta-lactam antibiotic production. Structure-based engineering of penicillin G acylase and cephalosporin acylase has resulted in improved enzymes for use in enzymatic production processes. The structures of many other enzymes that could be used in the production of beta-lactam antibiotics, such as enzymes from the beta-lactam biosynthetic pathway and beta-lactam antibiotic-converting enzymes, have been determined. The interest in these structures suggests that the future may see an even more extensive use of rationally engineered biocatalysts in antibiotic production than today.     

Biosynthesis of the beta-lactam antibiotic, thienamycin, by Streptomyces cattleya

Radioactive- and stable isotope-containing substrates were used to identify the biosynthetic precursors of the beta-lactam antibiotic, thienamycin, in Streptomyces cattleya. Acetate is utilized by the organism to form C(6) and C(7) of the beta-lactam ring. The two carbons of the hydroxyethyl group attached to C(6) are both derived from the methyl of methionine. The cysteaminyl side chain attached to C(2) is derived from cysteine. Selective inhibition of thienamycin and cephamycin C biosynthesis has been achieved either through the addition of metabolic inhibitors or through manipulation of the growth medium. These results suggest that the two beta-lactam antibiotics, thienamycin and cephamycin C, are formed by different biosynthetic pathways.     

Improved beta-lactam acylases and their use as industrial biocatalysts

Whereas the beta-lactam acylases are traditionally used for the hydrolytic processing of penicillin G and cephalosporin C, new and mutated acylases can be used for the hydrolysis of alternative fermentation products as well as for the synthesis of semisynthetic beta-lactam antibiotics. Three-dimensional structural analyses and site-directed mutagenesis studies have increased the understanding of the catalytic mechanism of these enzymes. The yield of hydrolysis and synthesis has been greatly improved by process design, including immobilization of the enzyme and the use of alternative reaction media. Significant advances have also been made in the resolution of racemic mixtures by means of stereoselective acylation/hydrolysis using beta-lactam acylases.     

Genetic engineering of beta-lactam antibiotic biosynthetic pathways in filamentous fungi.

Recombinant DNA technology has facilitated a rapid increase in our knowledge of beta-lactam antibiotic biosynthesis. Using the tools of this technology, beta-lactam biosynthetic genes and proteins have been characterized at the molecular level, cephalosporin-C production has been improved, new beta-lactams produced, and novel beta-lactam biosynthetic pathways have been constructed.