Specific β-lactam antibiotics inhibit secretion of lipo-β-lactamase in yeast

The β-lactam antibiotic cloxacillin can inhibit secretion of prokaryotic lipo-β-lactamase into the periplasm of yeast. The results indicate that this phenomenon is specific with respect to both the antibiotic and the lipo-β-lactamase whose secretion is affected, strongly suggesting that this involves an interaction between the enzyme and its substrates. The effect of the antibiotic on secretion is reversible. With different β-lactam antibiotics, the clearest difference is observed between type A and type S penicillins; the former exert a strong inhibition of secretion whereas the latter exhibit a weak effect or no effect at all. Type A penicillins have been previously shown to cause a conformational change in various β-lactamases. Mature lipo-β-lactamase species in yeast were localized either to the periplasmic space or bound to the outer surface of the cytoplasmic membrane and thus exposed to periplasm. The results are consistent with the hypothesis that binding of cloxacillin to lipo-β-lactamase induces a conformation on the protein that is unfavourable for its release from the membrane.     

Sensitivity of Escherichia coli to various beta-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic beta-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, beta-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic beta-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the beta-lactamases have been studied and their significance in creating beta-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of beta-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC beta-lactamase, or a plasmid-coded TEM-type beta-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter, 'target access index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.     

Analysis of bacterial carbapenem antibiotic production genes reveals a novel β-lactam biosynthesis pathway

Carbapenems are β-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora , makes the simple β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the β-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing β-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC , encode proteins which show significant homology with proteins encoded by the Streptomyces clavuligerus gene cluster responsible for the production of the β-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the β-lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β-lactam-ring-containing antibiotics.     

An active β-lactamase is a part of an orchestrated cell wall stress resistance network of Bacillus subtilis and related rhizosphere species

A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d -leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the β-lactamase PenP. This β-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to β-lactam resistance under different conditions. To test whether β-lactam resistance and β-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five β-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental β-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of β-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between β-lactams and β-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils.     

Cloning and expression in Enterobacteriaceae of the extended-spectrum β-lactamase gene from a Pseudomonas aeruginosa plasmid

Abstract An extended-spectrum β-lactamase, the gene for which is located on plasmid pMS350 in Pseudomonas aeruginosa strains, hydrolyzes carbapenems and other extended-spectrum β-lactam antibiotics. We cloned the pMS350 β-lactamase gene in an Escherichia coli K-12 strain using the vector plasmid pHSG398, and subcloned it into pMS360, a plasmid with a wide host-range. This resulted in the formation of the recombinant plasmid, pMS363, containing a 4.1-kb DNA insert that includes the extended-spectrum β-lactamase gene. Plasmid pMS363 was introduced into the P. aeruginosa PAO strain or into six species of Enterobacteriaceae, and the specific activities of the β-lactamase and MICs of various β-lactam antibiotics were estimated. The cloned gene was capable of expression in these strains and caused resistance to carbapenem, penem and other β-lactam antibiotics, with the exception of aztreonam.     

The effect of hydrophobicity of β-lactam antibiotics on their phospholipid bilayer permeability

Phospholipid bilayer permeability of β-lactam antibiotics was determined using liposomes enclosing β-lactamase. There was good correlation between the permeability and hydrophobicity within the analogous β-lactams. However, the effect of hydrophobic character on the permeability parameter was very different between the groups. Moderately hydrophilic penicillins such as benzylpenicillin and ampicillin showed very high permeability compared with cephalosporins. Penicillins having hindered side chains such as oxacillin and methicillin showed moderate permeability taking into account their hydrophobicity. These observations are suggestive of outer membrane permeation of these β-lactams via routes other than the porin pore, especially in porin-deficient mutants of gram-negative bacteria.     

Apparent variability in expression of the Bla+ phenotype of plasmid RP1 inPseudomonas acidovorans (RP1) strains

Expression of the Bla⁺ phenotype of the incompatibility group P-1 plasmid RP1 appears to be a variable phenomenon inPseudomonas acidovorans strains, although all plasmid-bearing strains examined synthesize the RP1-encoded TEM 2 β-lactamase. There is also evidence suggesting that plasmid-encoded alterations to the outer membrane could be affecting the degree of resistance observed to β-lactam drugs in at least some strains.     

Expression of pRW83 (penP) and pBR322-encoded β-lactamases in Escherichia coli

Abstract Plasmid pBR322 and penP -encoded β-lactamase activities were examined in cell fractions from wild-type and murein lipoprotein-deficient Escherichia coli strains. The specific activity of the Bacillus licheniformis penP gene product, a lipoprotein when expressed in E. coli , was increased in the outer membrane of a murein-lipoprotein deficient mutant. The activities of the 2 enzymes in wild-type E. coli exposed to the translational inhibitor puromycin were also investigated. Synthesis of penP was more susceptible to inhibition by puromycin than the pBR322-encoded TEM1 β-lactamase. The implications of these results for mechanisms of secretion and insertion of lipoproteins into the E. coli outer membrane are discussed.     

AmpD, essential for both β-lactamase regulation and cell wall recycling, is a novel cytosolic N-acetylmuramyl-L-alanine amidase

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of β-lactamase expression. It is shown here that the AmpD protein is a novel N-acetylmuramyl-L-alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptido-glycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a‘recycling tag’allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for β-lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.     

Characterization of β-lactamase activity using isothermal titration calorimetry

BackgroundHydrolysis of β-lactam antibiotic by β-lactamase is the most common mechanism of β-lactam resistance in clinical isolates. Timely detection and characterization of β-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on β-lactamases.MethodsA new assay was developed for the study of β-lactamase activity in protein solutions (Metallo-β-lactamase L1) and in clinical bacterial cells, based on heat-flow changes derived from enzymatic hydrolysis of β-lactams using isothermal titration calorimetry.Results(1) The thermokinetic parameters of three antibiotics (penicillin G, cefazolin and imipenem) and the inhibition constant of an azolylthioacetamide inhibitor were determined using the calorimetric assay. The results from the calorimetric assays were consistent with the data from the spectrophotometric assay. (2) The values of heat change in the calorimetric assay using two clinical Escherichia coli strains correlated well with their antibiotic susceptibility results from the broth dilution experiment. The subtypes of β-lactamase were also determined in the calorimetric assay.ConclusionsThe ITC assay is a reliable and fast method to study β-lactamase enzyme kinetics and inhibition. It can also provide thermodynamic information on antibiotic hydrolysis, which has been taken advantage of in this work to study β-lactamase activity in two clinical Escherichia coli isolates.General significanceAs the first calorimetric study of β-lactamase activity, it may provide a new assay to assist biomedical validation of new β-lactamase inhibitors, and also has potential applications on rapid antibiotic susceptibility testing and screening β-lactamase producing bacteria.     

The effects of βbT-lactams on the isoelectric focusing of βbT-lactamases

A range of concentrations of ceftazidime (4–64 mg I^-1) was shown to cause no induction of the TEM-1 and TEM-5 β-lactamases produced by Escherichia coli Nb. Increasing the concentration of ceftazidime in cultures of E. coli Nb caused a concomitant increase in the intensity of a satellite band of pI 5.2. The same increase in this satellite band was observed when ceftazidime was added to cell-free β-lactamase peparations from E. coli Nb and the separate addition of 11 different β-lactams to TEM-1 showed that each compound produced its own unique pattern of satellite bands. In addition, the mixing of ceftazidime with TEM-1 and 13 other TEM-derived β-lactamases caused a similar satellite band to be observed but ceftazidime did not have the same effect on PSE or SHV β-lactamases. Consequently, the addition of ceftazidime to a β-lactamase preparation prior to isoelectric focusing (IEF) may help to verify if a particular β-lactamase is TEM-derived. Purification of the satellite bands by electrodialysis and their subsequent re-focusing demonstrated that the ceftazidime-induced satellite bands can revert to a protein which has a pI similar to the parent band, illustrating the possible reversibility and dynamic nature of β-lactamase satellite bands on IEF. These results enable a better interpretation to be made of β-lactamase satellite bands observed on IEF.     

Resistance to Oxyimino β-Lactams Due to a Mutation of Chromosomal β-Lactamase in Citrobacter freundii

The duplicative mutation of an Ala-Val-Arg sequence at positions 208 to 210 in the loop structure of Enterobacter cloacae class C β-lactamase caused substrate specificity extension to oxyimino β-lactam antibiotics and this chromosomal mutation provided bacterial cells with high resistance to the β-lactams (M. Nukaga et al, 1995, J. Biol. Chem. 270, 5729-5735). In order to confirm the universality of this phenomenon among other class C β-lactamases, the duplicative mutation was applied to a class C β-lactamase of Citrobacter freundii, which has 74% homology to the E. cloacae β-lactamase amino acid sequence. The counterpart sequence to the Ala-Val-Arg of the E. cloacae enzyme in C. freundii β-lactamase was identified to be Pro-Val-His. A Pro-Val-His sequence was inserted just after the native Pro-Val-His sequence at positions 208 to 210 in the C. freundii β-lactamase. The resulting mutant of C. freundii β-lactamase obtained a striking characteristic that we expected, showing substrate specificity extension to oxyimino β-lactams. Nearly the same result was obtained with the insertion of an Ala-Val-Arg sequence after the native Pro-Val-His sequence. These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavorable substrates for many chromosomal class C β-lactamases produced by Gram-negative bacteria.     

Diazabicyclooctanes (DBOs): a potent new class of non-β-lactam β-lactamase inhibitors

The β-lactams have been among the most successful classes of antibacterial agents for the past half century. However, a disturbing increase in resistance to β-lactams has been noted among Gram-negative bacteria, which is attributable to β-lactamase enzymes not within the spectrum of currently marketed β-lactams or β-lactam/β-lactamase inhibitor combinations. Diazabicyclooctanes (DBOs) were first investigated as β-lactam mimics in the mid-1990s by chemists at Hoechst Marion Roussel (now part of Sanofi-Aventis) and proved to be a rich source of β-lactamase inhibitors (BLI). Two members of this novel series of highly potent, broad spectrum BLIs are now in clinical development and their properties are reviewed here.     

Co-operative binding of two Trp repressor dimers to α- or β-centred trp operators

The α-centred trp operator binds one dimer of the Trp repressor, whereas the β-centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr-Gly-7 substitution binds almost as well as the wild-type Trp repressor to the α-centred trp operator, but it does not bind to the β-centred trp operator. This confirms that Tyr-7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with a-centred trp operator variants showed that positions 1 of the a-centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the α-centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T-A to G-T) effectively transform the α-centred trp operator into a β-centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a β-centred trp operator.     

α3, β2, and β4 Form Heterotrimeric Neuronal Nicotinic Acetylcholine Receptors in Xenopus Oocytes

Abstract: One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different β subunits assemble with an α subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for α3, β2, and β4 subunits. β2 and β4, in pairwise combination with α3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). α3β4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through α3β2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for α3, β2, and β4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both β2 and β4 subunits in functional receptors.     

Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β-lactamase

In order to understand how TEM-1 β-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161–170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered β-lactamase enzymes indicated that while their catalytic efficiency (K_cat/K_m) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 β-lactamase.     

Complete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.     

Induction of penicillinase by bacitracin.

Bacitracin preparations are shown to induce penicillinase (β-lactamase I) formation in strain 569 of $\text{Bacillus cereus}$. At high bacitracin concentrations (2–4 mg/ml) the level of induced enzyme obtained reaches a maximum which is comparable to that induced by optimal concentrations (1–2 mcg/ml) of β-lactam antibiotics. Penicillinase formation induced by short exposure to bacitracin, continues at a normal rate after all free bacitracin has been removed. The inducing activity of bacitracin is highly, but not completely, resistant to β-lactamase and can be entirely eliminated by prolonged treatment with penicillinase of $\text{B. cereus}$. The site of induction by bacitracin is, however, different from that mediating induction by β-lactam antibiotics. The inducing component has been isolated by thin layer chromatography; it seems to be closely related to, but not identical with, bacitracin A,B or F.