Biomedical subjects

D McDevitt

Publications and source records attributed to D McDevitt.

At least 19 recordsLinked to original sources

PubMed2002

Novel targets for the future development of antibacterial agents.

Recent advances in DNA sequencing technology have made it possible to elucidate the entire genomes of pathogenic bacteria, and advancements in bioinformatic tools have driven comparative studies of these genome sequences. These evaluations are dramatically increasing our ability to make valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. In addition, developments in gene knockout technologies amenable to pathogenic organisms have enabled new genes and gene products critical to bacterial growth and pathogenicity to be uncovered at an unprecedented rate. Specific target examples in the areas of cell wall biosynthesis, aromatic amino acid biosynthesis, cell division, two component signal transduction, fatty acid biosynthesis, isopreniod biosynthesis and tRNA synthetases illustrate how aspects of the above capabilities are impacting on the discovery and characterization of novel antibacterial targets. An example of a novel inhibitor of bacterial fatty acid biosynthesis discovered from high throughput screening processes is described, along with its subsequent chemical optimization. Furthermore, the application and importance of technologies for tracking the mode of antibacterial action of these novel inhibitors is discussed.

Anti-Bacterial Agents↗

PubMed2002

Novel targets for the future development of antibacterial agents.

Animals↗

PubMed2001

The srhSR gene pair from Staphylococcus aureus: genomic and proteomic approaches to the identification and characterization of gene function.

Systematic analysis of the entire two-component signal transduction system (TCSTS) gene complement of Staphylococcus aureus revealed the presence of a putative TCSTS (designated SrhSR) which shares considerable homology with the ResDE His-Asp phospho-relay pair of Bacillus subtilis. Disruption of the srhSR gene pair resulted in a dramatic reduction in growth of the srhSR mutant, when cultured under anaerobic conditions, and a 3-log attenuation in growth when analyzed in the murine pyelonephritis model. To further understand the role of SrhSR, differential display two-dimensional gel electrophoresis was used to analyze the cell-free extracts derived from the srhSR mutant and the corresponding wild type. Proteins shown to be differentially regulated were identified by mass spectrometry in combination with protein database searching. An srhSR deletion led to changes in the expression of proteins involved in energy metabolism and other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology. The impaired growth of the mutant under anaerobic conditions and the dramatic changes in proteins involved in energy metabolism shed light on the mechanisms used by S. aureus to grow anaerobically and indicate that the staphylococcal SrhSR system plays an important role in the regulation of energy transduction in response to changes in oxygen availability. The combination of proteomics, bio-informatics, and microbial genetics employed here represents a powerful set of techniques which can be applied to the study of bacterial gene function.

Amino Acid Sequence↗

PubMed2001

Regulated ectopic expression and allelic-replacement mutagenesis as a method for gene essentiality testing in Staphylococcus aureus.

Conditional expression systems were utilized for the ectopic induction of essential genes in Staphylococcus aureus. Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and provided a direct and absolute confirmation of genetic essentiality for each locus. The procedure is particularly useful for genes that are difficult to analyze by conventional inactivation strategies due to either small size or complex genomic organization.

Alleles↗

PubMed2001

Exploiting genomics to discover new antibiotics.

There is an urgent need to develop new classes of antibiotics to tackle the increase in resistance in many common bacterial pathogens. One strategy to develop new antibiotics is to identify and exploit new molecular targets and this strategy is being driven by the wealth of new genome sequence information now available. Additionally, new technologies have been developed to validate new antibacterial targets, for example, new technologies have been developed to enable rapid determination of whether a gene is essential and to assess the transcription status of a putative target during infection. As a result, many novel validated targets have now been identified and for some, appropriate high-throughput screens against diverse compound collections have been carried out. Novel antibiotic leads are emerging from these genomics-derived targeted screens and the challenge now is to optimize and develop these leads to become part of the next generation of antibiotics.

Anti-Bacterial Agents↗

PubMed2001

A functional genomic analysis of type 3 Streptococcus pneumoniae virulence.

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature-tagged mutants was created by insertion-duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty-six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

Amino Acid Sequence↗

PubMed2000

Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action.

Selectively regulating gene expression in bacteria has provided an important tool for studying gene function. However, well-regulated gene control systems have been restricted primarily for use in laboratory non-pathogenic strains of bacteria (e.g. Escherichia coli, Bacillus subtilis). The development of analogous systems for use in bacterial pathogens such as Staphylococcus aureus would significantly enhance our ability to examine the contribution of any given gene product to pathogen growth and viability. In this report, we adapt, examine and compare three regulated gene expression systems in S. aureus, which had previously been used in B. subtilis. We demonstrate that all three systems function and exhibit titratable induction, together covering a dynamic range of gene expression of approximately 3000-fold. This dynamic range correlates well with the physiological expression levels of cellular proteins. Importantly, we show that one of these systems, the Spac system, is particularly useful for examining gene essentiality and creating specific conditional lethal phenotypes. Moreover, we find that titration of selective target gene products using this system allows direct demonstration of antibiotic mode of action.

Anti-Bacterial Agents↗

PubMed2000

The collagen-binding adhesin is a virulence factor in Staphylococcus aureus keratitis.

A collagen-binding strain of Staphylococcus aureus produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the cna gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of S. aureus infection of the cornea.

Adhesins, Bacterial↗

PubMed2000

The rel gene is essential for in vitro growth of Staphylococcus aureus.

The stringent response in Staphylococcus aureus is mediated by the nucleotide guanosine pentaphosphate, whose synthesis is catalyzed by the product of the rel gene. We report here that the rel gene is essential for the in vitro growth of S. aureus, distinguishing it from all other bacteria tested for this requirement.

Genes, Bacterial↗

PubMed2000

Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus.

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

Amino Acid Sequence↗

PubMed1999

Rapid method for the identification of essential genes in Staphylococcus aureus.

A strategy based on a vector host-dependent for autonomous replication, pSA3182, was utilized both for the rapid screening for Staphylococcus aureus genes essential for cell viability and for the introduction of specific polarity-neutral deletions in nonessential genes. The results obtained support the use of pSA3182 for both purposes.

Bacterial Proteins↗

PubMed1999

Isolation and characterization of a sigB deletion mutant of Staphylococcus aureus.

The sigB gene of Staphylococcus aureus, coding for the alternate sigma factor B, has been deleted by allelic replacement mutagenesis. The mutant grew as well as the parent in vitro, although it was deficient in clumping factor, coagulase, and pigment. In two murine and one rat infection model the mutant showed no reduction in virulence.

Animals↗

PubMed1998

The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site.

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin alphaIIbbeta3 recognize the same domain at the extreme C terminus of the fibrinogen gamma-chain. alphaIIbbeta3 interaction with this domain is known to occur in close proximity to a Ca2+-binding EF-hand structure in the alpha-subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca2+-binding EF-hand-like motif at residues 310-321 within the fibrinogen-binding domain. Deletion mutagenesis and site-directed mutagenesis of this EF-hand in recombinant truncated ClfA proteins (Clf40, residues 40-559; and Clf41, residues 221-559) resulted in a significant reduction of affinity for native fibrinogen and a fibrinogen gamma-chain peptide. Furthermore, Ca2+ (or Mn2+) could inhibit the binding of the fibrinogen gamma-chain peptide to Clf40-(40-559) and the adhesion of S. aureus cells to immobilized fibrinogen with an IC50 of 2-3 mM. In contrast, Mg2+ (or Na+) at similar concentrations had no effect on the ClfA-fibrinogen interaction. Far-UV CD analysis of Clf40-(40-559) and Clf41-(221-559) in the presence of metal ions indicated Ca2+- and Mn2+-induced differences in secondary structure. These data suggest that Ca2+ binds to an inhibitory site(s) within ClfA and induces a conformational change that is incompatible with binding to the C terminus of the gamma-chain of fibrinogen. Mutagenesis studies indicate that the Ca2+-dependent inhibitory site is located within the EF-hand motif at residues 310-321.

Adhesins, Bacterial↗

PubMed1997

Characterization of the interaction between the Staphylococcus aureus clumping factor (ClfA) and fibrinogen.

The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of biomaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-terminus of the fibrinogen gamma chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the gamma chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen gamma chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221-550) recognized intact recombinant fibrinogen gamma chains, but failed to recognize recombinant fibrinogen gamma chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen gamma chains contains a binding site for the integrin alphaIIb beta3 (glycoprotein gpIIb/IIIa) receptor on platelets [Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. & Hawiger, J. (1989) Biochemistry 28, 2915-1919; Farrell, D. H., Thiagarajan, P., Chung, D. W. & Davie, E. W. (1992) Proc. Natl. Acad. Sci. USA 89, 10729-10732; Hettasch, J. M., Bolyard, M. G. & Lord, S. T. (1992) Thromb. Haemostasis 68, 701-706]. We now show that Clf33 inhibits ADP-induced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 +/- 0.19 microM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal gamma-chain peptide effectively inhibited this interaction.

Adenosine Diphosphate↗

PubMed1997

Effect of quinupristin/dalfopristin on the outcome of vancomycin-resistant Enterococcus faecium bacteraemia: comparison with a control cohort.

Serious infection with vancomycin-resistant Enterococcus faecium (VREF) strains has no proven effective antimicrobial therapy. We compared the clinical and bacteriological outcomes of 20 patients with VREF bacteraemia treated with quinupristin/dalfopristin (RP 59500), an investigational streptogramin, with a historical cohort of 42 patients with VREF bacteraemia treated with other agents. Quinupristin/dalfopristin demonstrated in-vitro bacteriostatic activity against all 20 initial VREF blood isolates (MIC range 0.03-0.50 mg/L) by macrobroth dilution. The clinical characteristics of both groups were comparable for major outcome-dependent variables. There were five cases of recurrent VREF bacteraemia in the quinupristin/dalfopristin-treated cohort and 21 in the controls (P = 0.11); persistence of VREF at the primary site was found in six and 18 of the evaluable patients with follow-up cultures in these two cohorts (P = 0.06). In-hospital mortality was high in both groups: 65% in the quinupristin/dalfopristin group and 52% in the control group; however, VREF-associated mortality was significantly lower in the quinupristin/dalfopristin group (five and 17 respectively; P = 0.05). Follow-up susceptibility testing of five VREF isolates in the quinupristin/ dalfopristin group did not demonstrate resistance to quinupristin/dalfopristin. Quinupristin/ dalfopristin may be a useful agent for the therapy of serious VREF infection. Further clinical investigations are warranted to confirm or refute its clinical efficacy.

Adult↗

PubMed1997

Role of Staphylococcus aureus surface adhesins in orthopaedic device infections: are results model-dependent?

Bacterial colonisation of prosthetic material can lead to clinical infection or implant failure, or both, often requiring removal of the device. Adherence of Staphylococcus aureus to bioprosthetic materials is mediated by adhesins belonging to the MSCRAMM (microbial surface components recognising adhesive matrix molecules) family of microbial cell surface proteins. The objective of this study was to compare the virulence of a mutant strain of S. aureus Newman that possesses all three fibrinogen-, fibronectin- and collagen-binding MSCRAMMs (MSCRAMM-positive strain) with that of a mutant strain that lacks all three types of MSCRAMMs (MSCRAMM-negative strain) in a rabbit model of orthopaedic device-related infection. After a hole was drilled into the knee joint of each animal, a group of 10 rabbits was inoculated with the MSCRAMM-positive strain and another group of 10 rabbits received the MSCRAMM-negative strain. A stainless steel screw was then placed into the drilled hole. Two weeks later, the rabbits were killed and serum samples, bone tissue and implants were harvested for bacteriological and histopathological evaluation. No significant difference in infection rates was demonstrated between the two groups. The ability to delineate the role of S. aureus surface adhesins in causing orthopaedic device-related infection could be model-dependent.

Adhesins, Bacterial↗

PubMed1996

A low-fibronectin-binding mutant of Staphylococcus aureus 879R4S has Tn918 inserted into its single fnb gene.

A low-fibronectin-binding mutant of Staphylococcus aureus strain 879R4SSp generated by transposon Tn918 mutagenesis is attenuated in a rat endocarditis model (J.M. Kuypers & R.A. Procter, 1989, Infect Immun 57, 2306-2312). PCR and Southern hybridization analysis with primers and probes, respectively, for the fnbA and fnbB genes of strains 8325-4 showed that strain 879R4SSp possesses a single fnb gene which is homologous to fnbA. This was confirmed by sequencing 41 bp of 5' non-coding and 237 bp of 5' coding DNA, which showed 97% base identity to fnbA. Southern hybridization and sequencing showed that Tn918 was inserted 41 bp 5' to fnbA in the mutant 879R4SSp/1536, between the promoter and initiation codon. Reduced adherence of the mutant to surface-bound fibronectin correlated with lower expression of a 180 kDa wall-associated fibronectin-binding protein.

Adhesins, Bacterial↗

PubMed1996

Influence of agr on fibrinogen binding in Staphylococcus aureus Newman.

The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular catheters and host tissues during infection. We investigated the influence of the global regulator agr on the fibrinogen binding capacity and its relationship to the expression of coagulase (encoded by coa) and clumping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into strain Newman to obtain single, double, and triple mutants of the respective genes. As expected, the clfA mutant bound less soluble 125I-labeled fibrinogen than the corresponding coa mutant in agr+ strains; however, with agr mutant strains, the upregulation in fibrinogen binding capacity correlated mostly with the increased expression and transcription of coagulase as shown by Western (immunoblot) and Northern (RNA) blot analysis. In particular, the coa agr double mutant resulted in a significant reduction in fibrinogen binding compared with that of the agr mutant. The contribution of clfA to fibrinogen binding in agr-negative strains was less than that of coa (32,740 +/- 1,189 versus 18,141 +/- 334 and 38,919 +/- 1,021 cpm for clfA agr, coa agr, and the single agr mutant, respectively). Thus, coagulase is a major binding protein for soluble fibrinogen in the agr-negative background. In in vitro microtiter and catheter adherence assays with solid-phase fibrinogen, clumping factor, but not coagulase, plays a major role in binding to immobilized fibrinogen. coa transcription was negatively modulated by agr and occurred mainly during the exponential growth phase. In contrast, clfA transcription was agr independent and was strongest during the postexponential phase. Although an agr coa clfA triple mutant bound less soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 versus 18,141 +/- 334 cpm), significant residual fibrinogen binding capacity remained in the triple mutant, thus suggesting an additional fibrinogen binding component. By using direct ligand affinity blotting with 125I-fibrinogen, we could identify coagulase and an additional unidentified 52-kDa protein as a fibrinogen binding component in cell extracts. This band was absent in the extract of the coa clfA double mutant.

Bacterial Proteins↗