ABSTRACT
OBJECTIVES: Staphylococcus epidermidis biofilms form at the surface of implants and prostheses and are responsible for the failure of many antibiotic therapies. Only a few antibiotics are relatively active against biofilms, and rifampicin, a transcription inhibitor, is among the most effective molecules for treating biofilm-related infections. Having recently selected a new potential transcription inhibitor, we attempted to evaluate its efficacy against S. epidermidis biofilms. METHODS: Biofilm-forming S. epidermidis strains were grown planktonically or as biofilms and their susceptibility to this transcription inhibitor was compared with reference antibiotics with different mechanisms of action. CONCLUSIONS: Our results demonstrate that this new molecule is active; its effects are fast and kinetically related to those of rifampicin, but unlike rifampicin it does not select for resistant bacteria.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , DNA-Directed RNA Polymerases/antagonists & inhibitors , Rhodanine/analogs & derivatives , Rhodanine/pharmacology , Staphylococcus epidermidis/drug effects , Anti-Bacterial Agents/chemistry , Biofilms/growth & development , Humans , Microbial Sensitivity Tests , Polystyrenes , Rifampin/pharmacology , Staphylococcus epidermidis/growth & development , Transcription, Genetic/drug effectsABSTRACT
OBJECTIVES: Despite extensive functional screening of the bacterial RNA polymerase (RNAP) over the past years, very few novel inhibitors have been reported. We have, therefore, decided to screen with a radically different, non-enzymic, protein-protein interaction assay. Our target is the highly conserved RNAP-sigma interaction that is essential for transcription. METHODS: Small molecule inhibitors of the RNAP-sigma interaction were tested for their activity on transcription and on bacteria. RESULTS: These compounds have antibacterial activity against Gram-positive bacteria including multiresistant clinical isolates. CONCLUSIONS: This is, to our knowledge, the first example of a small molecule inhibitor of this interaction.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/enzymology , DNA-Directed RNA Polymerases/drug effects , Bacillus anthracis/drug effects , Bacillus cereus/drug effects , Drug Resistance, Bacterial , Enzyme-Linked Immunosorbent Assay , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Immunoprecipitation , Microbial Sensitivity Tests , Staphylococcus epidermidis/drug effects , Streptococcus pneumoniae/drug effects , Transcription, Genetic/drug effectsABSTRACT
We have recently isolated a monoclonal antibody directed against Escherichia coli RNA polymerase that does not inhibit transcription. This antibody is a useful tool to immobilize this enzyme for transcription assays or protein-protein interaction studies. The epitope of this monoclonal antibody was precisely located by a combination of protein deletion and synthetic peptide scanning. The amino acids of the epitope were also determined. We conclude that this antibody binds an epitope shared by several bacterial species and therefore can be used to characterize or purify other related enzymes.
Subject(s)
Antibodies, Monoclonal/metabolism , DNA-Directed RNA Polymerases/metabolism , Epitopes/genetics , Escherichia coli/enzymology , Amino Acid Sequence , Blotting, Western , Epitopes/metabolism , Molecular Sequence Data , Mutagenesis , Peptides/metabolism , Sequence Alignment , Surface Plasmon ResonanceABSTRACT
We have developed a multiwell assay for the detection of modulators of prokaryotic transcription based on the quantification of protein-protein interaction. This assay consists of three steps: (a) the immobilization of the Escherichia coli protein sigma70 in the well, (b) the incubation of the immobilized protein with core RNA polymerase and a potential inhibitor, and (c) washing and quantification of the binding of core to sigma70 with a monoclonal antibody conjugated to horseradish peroxidase. We show that this assay is sensitive, reproducible, and robust, and is able to discriminate between control competitors with different affinities. We demonstrate the usefulness of the assay to screen for microbial RNA polymerase inhibitors as potential new drugs for the treatment of emerging antibiotic-resistant bacteria.
