Fingerprinting of Bacterial Genomes by Amplification of DNA Fragments Surrounding Rare Restriction Sites

Masny, Aleksander; Płucienniczak, Andrzej

doi:10.2144/01314rr04

Cited by 15 publications

(15 citation statements)

References 6 publications

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“…Additional S. aureus clinical isolates were obtained from patients with skin infections. S. aureus molecular typing was performed using ADSRRS (amplification of DNA fragments surrounding rare restriction sites) fingerprinting analysis as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

Schmidtchen¹,

Pasupuleti²,

Mörgelin

et al. 2009

Journal of Biological Chemistry

127

105

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A novel approach for boosting antimicrobial peptides through end tagging with hydrophobic oligopeptide stretches is demonstrated. Focusing on two peptides derived from kininogen, GKHKNKGKKNGKHNGWK (GKH17) and HKHGHGH-GKHKNKGKKN (HKH17), tagging resulted in enhanced killing of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and fungal Candida albicans. Microbicidal potency increased with tag length, also in plasma, and was larger for Trp and Phe stretches than for aliphatic ones. The enhanced microbicidal effects correlated to a higher degree of bacterial wall rupture. Analogously, tagging promoted peptide binding to model phospholipid membranes and liposome rupture, particularly for anionic and cholesterol-void membranes. Tagged peptides displayed low toxicity, particularly in the presence of serum, and resisted degradation by human leukocyte elastase and by staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo and in vivo in porcine S. aureus skin infection models. The generality of end tagging for facile boosting of antimicrobial peptides without the need for post-synthesis modification was also demonstrated.

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Section: Methodsmentioning

confidence: 99%

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

Schmidtchen¹,

Pasupuleti²,

Mörgelin

et al. 2009

Journal of Biological Chemistry

127

105

View full text Add to dashboard Cite

show abstract

“…Additional S. aureus clinical isolates were obtained from patients with skin infections. S. aureus molecular typing was performed using ADSRRS-fingerprinting analysis (29). Briefly, S. aureus DNA was digested with the two restriction enzymes BamHI (10 units/µL) (Sigma) and XbaI (10 units/µL) (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Rational Design of Antimicrobial C3a Analogues with Enhanced Effects against Staphylococci Using an Integrated Structure and Function-Based Approach

et al. 2008

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The anaphylatoxin C3a and its inactivated derivative C3adesArg, generated during complement activation, exert direct antimicrobial effects, mediated via its C-terminal region [Nordahl et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 16879-16884]. During evolution, this region of C3a displays subtle changes in net charge, while preserving a moderate but variable amphipathicity [Pasupuleti et al. (2007) J. Biol. Chem. 282, 2520-2528]. In this study, we mimic these evolutionary changes, employing a design approach utilizing selected amino acid substitutions at strategic and structurally relevant positions in the original human C3a peptide CNYITELRRQHARASHLGLA, followed by structure-activity studies incorporating sequence-dependent QSAR models as tools for generation of C3a peptide variants with enhanced effects. While the native peptide and related amphipathic analogues of moderate positive net charge were active against the Gram-negative Escherichia coli, activity against the Gram-positive Staphylococcus aureus was primarily observed for peptides characterized by a combination of a relatively high net charge (+6-7) and a propensity to adopt an alpha-helical conformation with amphipathic character. Such increased helicity and charge also conferred activity against the fungus Candida albicans. A central histidine residue (H11), evolutionarily conserved among vertebrates, conferred high selectivity toward microbes, while substitutions with leucine rendered the peptides hemolytic. Selected C3a analogues retained their specificity against staphylococci in the presence of human plasma, while showing low cytotoxicity. The work illustrates structure-activity relationships underlying the function and specificity of antimicrobial C3a and related analogues and provides insights into the forces that drive evolution of antimicrobial peptides.

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“…In recent years, some alternative techniques have been successfully applied for the typing of bacteria below the species level. These include amplification-based methods, such as amplified fragment length polymorphisms (11), random amplification of polymorphic DNA (3,12,13), and amplification of DNA fragments surrounding rare restriction sites (5,6,8). These techniques are now applied more and more because they involve less time, comparably low costs, and only standard equipment.…”

mentioning

confidence: 99%

Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

et al. 2006

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In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of Escherichia coli isolates from patients of the Clinical Hospital in Gdańsk, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsedfield gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a hospital.Over the past 20 years, a significant number of DNA-based techniques have been introduced into the field of bacterial characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample. A variety of DNA fingerprinting techniques are presently available (1-7, 11-13), most of which use PCR for detection of fragments. The choice of fingerprinting technique depends on the type of application. Ideally, a fingerprinting technique should require no prior investments in terms of sequence analysis, primer synthesis, or characterization of DNA probes. A number of fingerprinting methods which meet these requirements have been developed. The fingerprints are obtained by visualizing many parts of the genome. Differences in these fingerprints between individuals are interpreted as genetic distances. Obviously, the differences should reflect variations in DNA rather than artifacts due to a nonrobust method. Furthermore, the method should provide the appropriate level of discriminatory power, and it should be relatively rapid and cheap, especially in large-scale population genetic studies. Macrorestriction analysis of genomic DNA followed by pulsed-field gel electrophoresis (REA-PFGE) has become the "gold standard" for molecular typing (10). In recent years, some alternative techniques have been successfully applied for the typing of bacteria below the species level. These include amplification-based methods, such as amplified fragment length polymorphisms (11), random amplification of polymorphic DNA (3, 12, 13), and amplification of DNA fragments surrounding rare restriction sites (5, 6, 8). These techniques are now applied more and more because they involve less time, comparably low cos...

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Fingerprinting of Bacterial Genomes by Amplification of DNA Fragments Surrounding Rare Restriction Sites

Cited by 15 publications

References 6 publications

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

Rational Design of Antimicrobial C3a Analogues with Enhanced Effects against Staphylococci Using an Integrated Structure and Function-Based Approach

Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

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