By contrast, carolacton is structurally unrelated to peptide pheromones. check details Proof of principle for using chemically unrelated compounds as inhibitors has been obtained for the acylated homoserine lactone based quorum sensing system of Gram negative bacteria [55]. Conclusions Bacterial signalling systems have emerged in recent years as attractive targets for antimicrobial therapy. The discovery of
a compound damaging S. mutans biofilms which might be targeting one or several of its two-component systems involved in regulating biofilm formation, autolysis and stress tolerance could provide a novel approach for future therapeutic strategies to prevent dental plaque related diseases with only minimal impact see more on the normal microbial flora. Methods Bacterial strains and culture conditions S. mutans wild-type strain UA159 (ATCC 700610) and its knockout Luminespib chemical structure mutants defective in the quorum sensing genes comC, comD, or comE have been provided by courtesy of Prof. Dr. D. G. Cvitkovitch from the University of Toronto, Canada. The mutants were constructed by allelic replacement of the gene in question with an erythromycin resistance cassette using the PCR ligation mutagenesis strategy described in more detail in [56]. The wild-type strain was maintained routinely on Todd-Hewitt (TH) agar plates (Difco) and liquid
cultures were grown in Todd-Hewitt broth Bacto™(THB). For cultivation of the mutants, erythromycin was added at 10 μg per ml to the media. For biofilm growth, THB was supplemented with 0.5% sucrose (THBS). Incubation was at 37°C without agitation under aerobic (with 10% CO2) or anaerobic (80% N2, 10% H2, 10% CO2) conditions. For anaerobic growth, the medium was flushed with nitrogen before use. Escherichia coli DH5α was used as cloning strain and routinely cultured in Luria Bertani (LB, Carl-Roth, Karlsruhe, Germany) medium at 37°C. E. coli strains carrying plasmids were selected with 50 μg ml-1 spectinomycin. Inhibition of planktonic growth and determination of cytotoxicity The minimal inhibitory concentration of carolacton
on planktonic growth of S. mutans UA159 was determined with the conventional serial two-fold dilution method in 96-well microtiter plates (200 μl/well). As inoculum 1 × 106 cells/ml were used, and carolacton was dissolved in MeOH, producing concentrations Unoprostone in the cultures of not more than 5%. Incubation was for 24 hours at 37°C under both anaerobic and aerobic conditions. Optical density (OD) measurements at 620 nm were performed using a Wallac Victor3™1420 Multilabel Counter (Perkin-Elmer Life Sciences). Acute cytotoxicity against L929 mouse cells (connective tissue, ATCC CCL 1) was determined using an MTT assay as reported [57]. Cytoplasmic histone-associatd DNA fragments were measured with the Cell Death Detection ELISA kit from Roche Diagnostic to determine apoptosis induction in L929 cells.