Biomedical subjects

R C Levesque

Publications and source records attributed to R C Levesque.

At least 19 recordsLinked to original sources

PubMed2001

In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa.

Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.

Amino Acid Sequence↗

PubMed2001

Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies.

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.

Alanine↗

PubMed2001

Discovering essential and infection-related genes.

Transposon-based approaches are very powerful for identification of essential and infection-related genes in bacteria, particularly in the context of microbial genomics. We describe recent progress in several of these approaches, and their underlying principles. The essential gene test (EGT) is a transposon-based technique that can rapidly identify a nucleotide sequence from a database as essential or dispensable. Also, variations of in vitro transposon mutagenesis applications, such as genomic analysis and mapping by in vitro transposition (GAMBIT), are described. The development of techniques including PCR-based signature-tagged mutagenesis is now used to find essential virulence genes in different bacterial hosts. These approaches form the basis for the identification of microbial targets in development of novel antimicrobials and vaccines by the biotechnology and pharmaceutical industry.

Bacteria↗

PubMed2000

Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa.

Novel putative pyoverdine synthetase pvdIJK genes were found upstream of pvdD in the 6.2-Mb chromosome of Pseudomonas aerugilosa strain PAO1. These genes formed a locus implicated in pyoverdine biosynthesis. Sequence analysis showed that the product of these genes shared 43%, 60% and 57% identity with PvdD. PvdIJK are thought to be implicated in synthesis of pyoverdine, a siderophore chelating Fe3+. A pvdI mutant was obtained by gene disruption mutagenesis and confirmed by Southern hybridization. The pvdl mutant produced gave no significant growth on solid media supplemented with the iron chelator 2,2-dipyridyl; while the PvdI- phenotype abolished pyoverdine fluorescence. The role of PvdI in pathogenicity was tested by measuring the in vivo growth of P. aeruginosa wild-type and mutant strains in a chronic lung infection rat model, and by measuring the competitive infectivity index into a neutropenic mice model. The data obtained confirmed the importance of PvdI in virulence and iron uptake.

Amino Acid Sequence↗

PubMed2000

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors.

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.

Acylation↗

PubMed2000

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Amino Acid Sequence↗

PubMed2000

Detection of genes essential in specific niches by signature-tagged mutagenesis.

Variations of the signature-tagged mutagenesis (STM) technique are now possible and the method can be applied to most pathogens that have an STM-selectable phenotype in a host system. STM screening of 15,040 mutants from 11 bacterial species identified 323 in vivo attenuated mutants. As a genome-scanning tool, STM will yield information about genes with unknown functions as well as information crucial for understanding microbial pathogenesis.

Animals↗

PubMed2000

Combinatorial biochemistry and shuffling of TEM, SHV and Streptomyces albus omega loops in PSE-4 class A beta-lactamase.

The class A PSE-4 beta-lactamase was used for studying the importance of amino acids in the omega (Omega) loop and its interactions for hydrolysis of beta-lactam antibiotics. By cassette mutagenesis, we replaced the amino acids 163-179 Omega loop in PSE-4 with TEM-1, SHV-1 and Streptomyces albus G beta-lactamase Omega loops. Phenotypic analysis of Escherichia coli recombinants expressing the Omega loop PSE-4 mutant enzymes gave MICs and kinetic data similar to those of wild-type PSE-4.

Amino Acid Substitution↗

PubMed2000

Structure-function analysis of alpha-helix H4 using PSE-4 as a model enzyme representative of class A beta-lactamases.

We extracted maximum information for structure-function analysis of the PSE-4 class A beta-lactamase by random replacement mutagenesis of three contiguous codons in the H4 alpha-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structure-function studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 alpha-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face pi-pi interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 alpha-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 alpha-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 alpha-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.

Circular Dichroism↗

PubMed2000

Molecular basis of antibiotic resistance and beta-lactamase inhibition by mechanism-based inactivators: perspectives and future directions.

Antibacterial chemotherapy is particularly striking in the family of penicillins and cephalosporins. Over 40 structurally different beta-lactam molecules are available in 73 formulations and the majority of them are currently prescribed for medical use in hospitals. beta-Lactams are well tolerated by humans with few side effects. They interact very specifically with their bacterial target, the D-alanyl-D-alanine carboxypeptidase-transpeptidase usually referred to as DD-peptidase. The outstanding number of beta-lactamases produced by bacteria represent a serious threat to the clinical utility of beta-lactams. The discovery of beta-lactamase inhibitors was thought to solve, in part, the problem of resistance. Unfortunately, bacteria have evolved new mechanisms of resistance to overcome the inhibitory effects of beta-lactamase inactivators. Here, we summarize the diversified mechanistic features of class A beta-lactamases interactions with mechanism-based inhibitors using available microbiological, kinetic and structural data for the prototype TEM beta-lactamases. A brief historical overview of the strategies developed to counteract beta-lactamases will be presented followed by a short description of the chemical events which lead to the inactivation of TEM beta-lactamase by inhibitors from different classes. Finally, an update on the clinical prevalence of natural and inhibitor-resistant enzyme mutants, the total chemical synthesis to design and synthesize a new structure and produced a broad spectrum beta-lactamase inhibitor that mimics the beta-lactam ring, but does not contain it is discussed.

Anti-Bacterial Agents↗

PubMed2000

In vivo-induced genes in Pseudomonas aeruginosa.

In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).

Animals↗

PubMed1999

Strategies for isolation of in vivo expressed genes from bacteria.

The discovery and characterization of genes specifically induced in vivo upon infection and/or at a specific stage of the infection will be the next phase in studying bacterial virulence at the molecular level. Genes isolated are most likely to encode virulence-associated factors or products essential for survival, bacterial cell division and multiplication in situ. Identification of these genes is expected to provide new means to prevent infection, new targets for, antimicrobial therapy, as well as new insights into the infection process. Analysis of genes and their sequences initially discovered as in vivo induced may now be revealed by functional and comparative genomics. The new field of virulence genomics and their clustering as pathogenicity islands makes feasible their in-depth analysis. Application of new technologies such as in vivo expression technologies, signature-tagged mutagenesis, differential fluorescence induction, differential display using polymerase chain reaction coupled to bacterial genomics is expected to provide a strong basis for studying in vivo induced genes, and a better understanding of bacterial pathogenicity in vivo. This review presents technologies for characterization of genes expressed in vivo.

Cloning, Molecular↗

PubMed1999

Structure-function studies of Ser-289 in the class C beta-lactamase from Enterobacter cloacae P99.

Site-directed mutagenesis of Ser-289 of the class C beta-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in beta-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant beta-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC beta-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five beta-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant beta-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

Bacterial Proteins↗

PubMed1999

Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria.

We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.

Base Sequence↗

PubMed1998

Pseudomonas aeruginosa PAO1 bacterial artificial chromosomes: strategies for mapping, screening, and sequencing 100 kb loci of the 5.9 Mb genome.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen frequently found in nosocomial infections and is a major cause of morbidity and mortality in patients with cystic fibrosis. To facilitate molecular studies of this organism, we have generated a bacterial artificial chromosome (BAC) library. Genomic DNA was isolated from the prototype strain PAO1, partially digested with HindIII, size selected after pulsed-field gel electrophoresis, and used to construct a BAC library using the pBeloBAC11 vector. DNAs from approximately 850 clones, representing more than 9.5-fold physical coverage of the 5.9-Mb PAO1 genome, were analyzed after SpeI and HindIII digestions and agarose gel electrophoresis. The BAC library had clones with insert fragments ranging from 20 to more than 290 kb. A subset of 264 BACs having inserts > 80 kb, representing > 4 genome equivalents, were rearrayed into 96-well plates, and a clone pooling and PCR screening strategy was developed. The PCR library screening enabled the identification and recovery of BACs containing genes implicated in cell division and in cell wall biosynthesis, as well as a series of known genes mapping to different regions of the PAO1 chromosome. A physical and genetic map was constructed for the 98-kb pMOC5 BAC clone, which spans the entire fts-mur locus. Chromosome walking from each end of the pMOC5 clone placed it within a contig spanning 243 kb. The BAC library and screening resources now allow a PCR-based screening of a P. aeruginosa genomic library for any gene of interest. The restriction fragment analysis of overlapping clones indicated that BAC clones stably maintain and propagate Pseudomonas DNA, providing evidence that the PAO1 BAC library is an appropriate reagent for genome sequencing.

Bacterial Proteins↗

PubMed1998

Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime.

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Omega loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Omega loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

Amino Acid Sequence↗

PubMed1998

Molecular heterogeneity of the L-1 metallo-beta-lactamase family from Stenotrophomonas maltophilia.

We have determined the nucleotide sequence of the blaS gene encoding the carbapenem-hydrolyzing L-1 beta-lactamase from Stenotrophomonas maltophilia GN12873. Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids. Comparisons of the L-1 amino acid sequence with those of other zinc beta-lactamases showed 88.6% identity with the L-1 enzyme from S. maltophilia IID1275 and less than 20% identity with other class B metalloenzymes.

Amino Acid Sequence↗

PubMed1998

Structure of CARB-4 and AER-1 carbenicillin-hydrolyzing beta-lactamases.

We determined the nucleotide sequences of blaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing beta-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these beta-lactamases varied from 98.7 to 99.9%, while that between these genes and blaCARB-4 encoding CARB-4 was 86.3%. The blaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 beta-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A beta-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking blaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.

Amino Acid Sequence↗