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.     

Insertion of dibasic residues directs a constitutive protein to the regulated secretory pathway.

The mechanisms for sorting proteins to the regulated secretory pathway (RSP) remains poorly understood. We recently reported that dibasic sequences that are cleaved by pro-protein convertases (PCs) in pro-neurotensin also acted as sorting signal for the precursor. Here we addressed two questions regarding the role of dibasics as sorting signal: (i) Are dibasics sufficient to direct proteins to the RSP? (ii) Do they sort proteins by virtue of their interaction with PCs? The first question was studied by inserting dibasics in beta-lactamase, a constitutively secreted protein and comparing the regulated secretion of beta-lactamase to that of its mutant in transfected endocrine cells. The second question was investigated by comparing the regulated release of pro-neurotensin in PC12 cells that are devoid of PCs to that in PC1- and PC2-transfected PC12 cells. The data show that the mutant beta-lactamase was indeed targeted in part to the RSP and that pro-neurotensin was sorted to the RSP without the assistance of the PCs, thus indicating that dibasics can act as sorting signal by themselves independently of their interaction with PCs.     

Nucleotide sequence of the thermostable direct hemolysin gene (tdh gene) of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio parahaemolyticus

During an outbreak of infection with ampicillin-resistant, TEM-1 beta-lactamase-producing Escherichia coli serotype O15, some strains were noted to differ from the majority in that they showed reduced susceptibility to amoxycillin/clavulanic acid (Augmentin), ureidopenicillins and first generation cephalosporins and produced increased amounts of beta-lactamase. The plasmid from one such isolate was compared with that from an isolate that produced normal amounts of beta-lactamase. Restriction analysis with EcoRI revealed extra fragments in the plasmid from the beta-lactamase hyperproducer and use of DNA-DNA hybridisation with a biotinylated TEM-1 probe showed genetic rearrangement in the beta-lactamase hyperproducer so that the TEM gene appeared to be present in larger amounts and was located on a smaller fragment than for the plasmid from the strain that produced normal amounts of beta-lactamase.     

Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response

The intensive antibiotic treatment of cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa has improved the survival rate and the clinical condition of Danish patients. Acquirement of resistance to anti-pseudomonal antibiotics is one of the main drawbacks of this therapeutic strategy and our results showed the development of resistance of P. aeruginosa to several antibiotics during 25 years of intensive antibiotic treatment. Our studies have been concentrating on the development of resistance to beta-lactam antibiotics. We have shown an association between the development of resistance to beta-lactam antibiotics and the occurrence of high beta-lactamase producing strains and between the MIC of the beta-lactams and the levels of beta-lactamase expression. Partially derepressed mutants, characterized by high basal levels of beta-lactamase with the possibility of induction to even higher levels during treatment with beta-lactam antibiotics, were the most frequent phenotype found among resistant Danish P. aeruginosa CF isolates. We have also shown that the high alginate producing P. aeruginosa isolates, that characterize the chronic lung infection in CF patients, are more susceptible to antibiotics and produce less beta-lactamase than the non-mucoid paired isolates. We propose that the non-mucoid isolates are exposed to a relatively higher antibiotic pressure than the mucoid isolates and therefore, they become easily antibiotic resistant and in consequence produce high levels of beta-lactamase. The beta-lactamase produced by the non-mucoid isolates might play a protective role in the biofilm, defending the mucoid isolates from the action of beta-lactam antibiotics and helping them to maintain their antibiotic susceptibility. We have also shown that beta-lactamase, which is a periplasmic enzyme, can be secreted extracellulary packed in membrane vesicles liberated by high beta-lactamase-producing P. aeruginosa. The continuos presence in the CF lungs of bacteria producing high basal levels of beta-lactamase (partial derepressed) induces a humoral immune response to beta-lactamase. We have shown that antibodies against the chromosomally encoded beta-lactamase (a beta ab) might be considered a marker of the development of resistance to beta-lactam antibiotics. We investigated the humoral immune response to beta-lactamase by quantifying a beta ab specific IgG and IgG subclass antibodies, by investigating the influence of the allotypes on the IgG subclass response and by measuring the avidity of the IgG a beta ab. We found that CF patients with good lung function had in the early stages of the chronic lung infection higher titers of a beta ab of good avidity than patients with poor lung function. Therefore, we raised the hypothesis that some of the a beta ab might have beta-lactamase neutralizing effect, playing a beta-lactamase inhibitor role and improving the effect of the treatment with beta-lactam antibiotics. Finally, we tested our hypothesis in the rat model of chronic lung infection by assessing the effect of a beta ab raised by vaccination with purified chromosomal beta-lactamase on the outcome of the treatment with ceftazidime of bacteria resistant to beta-lactam antibiotics. Our results showed that significantly lower bacterial load and better lung pathology were found in rats with neutralizing antibodies compared to non-immunized rats or rats without neutralizing antibodies. Our findings might be of potential importance for the improvement of the treatment with beta-lactam antibiotics of resistant P. aeruginosa hyperproducing chromosomal beta-lactamase that represent a threat especially for patients with CF and chronic lung infection.     

Disulphide bridge formation in the periplasm of Escherichia coli: β-lactamase::human lgG3 hinge fusions as a model system

We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.     

Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus

The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

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.     

Design, synthesis, and evaluation of alpha-ketoheterocycles as class C beta-lactamase inhibitors

A series of specific alpha-ketoheterocycles (benzoxazole, thiazole, imidazole, tetrazole, and thiazole-4-carboxylate) has been synthesized in order to assess their potential as beta-lactamase inhibitors. The syntheses were achieved either by construction of the heterocycle (benzoxazole) from an appropriate alpha-hydroxyimidate, followed by oxidation of the alcohol, or by direct reaction of methyl phenaceturate with a lithiated heterocycle. The properties of these compounds in aqueous solution are described and their inhibitory activity against beta-lactamases assessed. They did inhibit the class C beta-lactamase of Enterobacter cloacae P99 but not the TEM beta-lactamase. The most effective inhibitor of the former enzyme (K(i)=0.11 mM) was 5-(phenylacetylglycyl) tetrazole, probably because it is an anion at neutral pH. Interpretation of the results was aided by computational models of the tetrahedral adducts. Most of the compounds also inhibited alpha-chymotrypsin but not porcine pancreatic elastase.     

Group 1 beta-lactamases of Aeromonas caviae and their resistance to beta-lactam antibiotics

The contribution of beta-lactamase production to beta-lactam antibiotic resistance was examined in an Aeromonas caviae mutant strain, selected in vitro by cefotaxime and derived from a wild-type strain isolated in our laboratory from crude sewage. Both strains produced beta-lactamase. The mutant strain (AC7m) produced beta-lactamase constitutively, in contrast to the parental strain (AC7), which was inducible by cefoxitin. AC7m was regarded as a mutant from AC7, which over-expressed beta-lactamase. The mutant strain showed a remarkable reduction in sensitivity to most of the beta-lactam antibiotics tested, such as (i) aminopenicillins and their combinations with clavulanic acid and sulbactam, (ii) carboxypenicillins, (iii) ureidopenicillins, and (iv) cephalosporins. This strain remained susceptible to ceftazidime, imipenem, and aztreonam. Isoelectric focusing of sonic extracts revealed that both strains AC7 and AC7m shared a common major beta-lactamase band at pI 6.5. The plasmid DNA assays showed that the beta-lactamases expressed by each A. caviae strain were chromosomally encoded. Based on substrate and inhibitor profiles determined in sonic extracts for AC7 and AC7m, the enzymes displayed on isoelectric focusing at pI 6.5 were assigned to chromosomal Group 1 beta-lactamases. Imipenem would therefore be the appropriate choice for therapy of infections caused by A. caviae beta-lactamase over-expressing mutants.     

Signalling proteins in enterobacterial AmpC β-lactamase regulation

The cloned Citrobacter freundii ampC beta-lactamase is inducible in the presence of its regulatory gene ampR in Escherichia coli (Lindberg et al., 1985). The basal level of expression and inducibility are affected by two E. coli proteins encoded by the closely linked ampD and ampE genes. Deletion of both genes led to constitutive ampR-dependent overproduction of beta-lactamase, whereas an out-of-frame deletion in AmpD caused the basal expression to increase two-fold. This ampD1 mutant was inducible at lower beta-lactam concentrations than the wild type. An IS1 insertion in ampD was polar on ampE expression and increased basal beta-lactamase expression 30-fold while mediating a semi-constitutive phenotype. AmpE expressed from a recombinant plasmid in an ampD-ampE deletion mutant reduced basal beta-lactamase expression to wild-type levels but did not markedly reduce beta-lactam resistance since the cells became hyperinducible. In the absence of AmpD, increasing levels of AmpE therefore decrease the basal expression of AmpC beta-lactamase in an AmpR-dependent manner. AmpD modulated the response exerted on beta-lactamase expression by AmpE. The ampD gene encodes a 20.5kD cytoplasmic protein while the 32.1kD ampE gene product is an integral membrane protein with a likely ATP-binding site between the second and third putative transmembrane region. Since neither AmpD nor AmpE are needed for beta-lactam induction and since these proteins could not be covalently labelled by benzylpenicillin, they are not thought to act as beta-lactam-binding sensory transducers. Instead it is suggested that AmpD and AmpE sense the effect of beta-lactam action on peptidoglycan biosynthesis and relay this signal to AmpR.     

Prediction of two- and three-amino-acid sequences of Citrobacter Freundii beta-lactamase from its amino acid composition

The repeated amino-acid sequences in Citrobacter Freundii beta-lactamase may be indispensable for its function, because such repetitions cannot be simply attributed to a chance. In order to fully explore the functional units in Citrobacter Freundii beta-lactamase, it may need to analyse all the amino acid pairs, triplets, etc. along Citrobacter Freundii beta-lactamase from one terminal to the other terminal, to count their frequencies and calculate their probabilities. The amino-acid sequence of Citrobacter Freundii beta-lactamase was counted according to two-, three- and four-amino-acid sequences. The counted frequency and probability were compared with the predicted frequency and probability. The amino acid sequences, which appear in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately evolved and conserved. By contrast, the amino acid sequences, which appear in Citrobacter Freundii beta-lactamase but cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately evolved and conversed. Accordingly 99 (26.053%) and 33 (8.684%) of 380 two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism. Some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately excluded from Citrobacter Freundii beta-lactamase. By contrast, some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately excluded from Citrobacter Freundii beta-lactamase. Accordingly 89 (48.370%) and 41 (22.283%) of 184 kinds of absent two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism, and 7236 (99.848%) of 7247 kinds of absent three-amino-acid sequences can be predicted by the frequency according to a purely random mechanism. The amino acids, whose probabilities in following certain preceding amino acids can be predicted from Citrobacter Freundii beta-lactamase amino acid composition according to a purely random mechanism, should not be deliberately evolved and conversed, accordingly 2 (0.526%) of 380 counted first order Markov transition probabilities for the second amino acid in two-amino-acid sequences match the predicted conditional probabilities.     

Identification of amino acid sequences that can function as translocators of β-lactamase in Escherichia coli

A plasmid vector, pYZ1, was constructed which lacks most of the beta-lactamase signal-peptide coding region, but has a unique EcoRI site spanning codons 2 and 3 of the resultant cytoplasmic beta-lactamase derivative. Short quasi-random DNA sequences were cloned into the EcoRI site and Escherichia coli transformants in which some translocation of beta-lactamase across the cytoplasmic membrane was restored were selected by their ability to survive and form colonies on plates containing a low level of ampicillin. About 15-20% of all in-frame inserts restored some beta-lactamase translocation and the salient feature of these sequences was their marked hydrophobicity. These results are discussed in the light of a similar study in which sequences able to function as translocators of invertase in yeast were cloned and analysed (Kaiser et al., 1987).     

Secretion of beta-lactamase requires the carboxy end of the protein

Synthesis and secretion of beta-lactamase were studied in Salmonella typhimurium infected with P22 phage carrying the structural gene for beta-lactamase (the bla gene) in mutant or wild-type form. The wild-type gene was shown to specify two forms of beta-lactamase which differ in molecular weight by about 2500 daltons. This difference is consistent with removal, predicted on other grounds, of 23 amino-terminal residues (the "signal" sequence). All bla- mutants, including chain-terminating mutants lacking as much as 50% or as little as 10% of the protein, were apparently unaffected in this processing step. Pulse-chase experiments showed that more than 85% of the wild-type (as well as mutant) proteins are synthesized as complete overlength precursors before being processed to their mature forms. Virtually all the mature wild-type protein appears in the periplasmic space whereas a large fraction of the precursor appears in the cytoplasm. In contrast, both the precursor and processed forms of beta-lactamase proteins synthesized by chain-terminating mutants (including one which lacks only 10% of its residues from the carboxy end) are not secreted and apparently remain soluble in the cytoplasm. These results show that the carboxy-terminal amino acid sequence (at least) of beta-lactamase is essential to successful transport across the cytoplasmic membrane, and suggest that the presence (and probably also the act of removal) of the signal sequence does not suffice to ensure secretion.     

Expression of beta-lactamase in Coxiella burnetii transformants

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.     

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.     

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.     

Characterization of the membrane beta-lactamase in Bacillus cereus 569/H/9

The membrane-bound beta-lactamase from Bacillus cereus, strain 569/H/9, has been purified to apparent homogeneity. Nonionic detergent (0.5% Triton X-100) is required to keep the enzyme (traditionally called gamma-penicillinase and now called beta-lactamase III) in solution. Antibodies to beta-lactamase III have been prepared, and the membrane-bound enzyme is immunochemically distinct from the extracellular enzymes. beta-Lactamase III has a molecular weight of 31 500, in contrast to the extracellular enzymes beta-lactamase I and beta-lactamase II which have molecular weights of 30 000 and 22 000, respectively. The isoelectric point of beta-lactamase III is pH 6.8, whereas beta-lactamase I and beta-lactamase II have isoelectric points about 8.6 and 8.3. The amino acid composition of beta-lactamase III differs from those of beta-lactamase I and beta-lactamase II; however, the difference index between the compositions of beta-lactamase I and beta-lactamase III (52%) suggests relatedness. beta-Lactamase III is inactivated by 6 beta-bromopenicillanic acid and by the sulfone of 6 alpha-chloropenicillanic acid, and cephalosporins are poorer substrates than penicillins. beta-Lactamase III may be a membrane-bound class A beta-lactamase.