Project name: Streptococcus pneumoniae D39

Description: Streptococcus pneumoniae is a Gram positive bacterium that causes severe invasive infection such as pneumonia, septicemia, meningitis and otitis media especially in children, the elderly and immune-compromised patients. Pneumococcal colonization and disease is often associated with biofilm formation. Bacteria in biofilms exhibit elevated resistance both to antibiotics and to host defense systems, which often results in persistent and difficult-to-treat infections. Therefore, the ongoing treat to human health posed by pneumococcal biofilms has prompted extensive research aimed to identify alternative targets and new antimicrobial agents that are effective against bacteria biofilms. The effective anti-biofilm strategies should include inhibition of microbial adhesion to the surface and of colonization, interference with the signal molecules modulating biofilm development and the disaggregation of the biofilm matrix.In this study, we examine the effect of DAM inhibitor small molecule pyrimidine-diones on streptococcus pneumoniae D-39 strain growth (planktonic and biofilm) and evaluate the changes in global gene expression using c-DNA microarray. The microarray analysis was performed on total RNA extracted from biofilms grown in 24-well microtiter plate with 7µm/ml pyrimidine-diones small molecule and control biofilms (biofilms grown without pyrimidine-diones small molecule). To validate the results of microarray, real-time RT-PCR was performed on 12 differentially expressed genes from six different functional groups. cDNA-microarray analysis detected a total of 259 genes that were significantly differentially expressed in biofilm growth with pyrimidine-diones small molecule. 204 genes were significantly down expressed and 55 genes were significantly up expressed in biofilms grown with 7µm/ml pyrimidine-diones small molecule. Among the 204 down expressed genes, 45 were hypothetical protein encoding gene and 159 were functional protein encoding genes. Of 55 up-regulated genes 21 were hypothetical genes and 34 were functional protein encoding genes. The functional annotation showed that gene involve in fatty acid metabolism, cell division, cell cycles, DNA metabolism, cell assembly were significantly down regulated and galactose metabolism related gene were up-expressed in biofilm grown with pyrimidine-diones small molecule.Overall design: S. pneumoniae D-39 strain (NCTC 7466) is an encapsulated, serotype 2 pathogenic strain. It was obtained from the Health Protection Agency Culture Collections (HPA, Salisbury, UK). Bacteria were routinely grown in tryptic soy broth (TSB; BD Difco, Detroit, MI, USA) or on blood agar (BA; Hye In, Seoul, Korea) supplemented with 5 % v/v sheep blood at 37 °C in an atmosphere of 5% CO2. Small molecule pyrimidine-diones with chemical name 1-(4 bromophenyl)-5-(2-furylmethylene)-3-phenyl-2-thioxodihydro-4, 6 (1H,5H)-pyrimidinedione was purchased from Chembridge, USA. Stock solution of small molecule pyrimidine-diones was prepared in DMSO.In vitro biofilm formation was carried out in 24-well, flat-bottom, polystyrene microtiter plate (BD falcon, MD, USA) in static model. S. pneumoniae grown up to mid-logarithmic phase in TSB medium was diluted 1:100 with fresh sterile TSB medium, inoculated 1.5 mL in 24-well microtiter plate and, incubated for 15 hours at 37°C in 5% CO2. Pyrimidine-diones small molecule was supplied at the concentration of 7µm/ml. Control biofilm were supplied with equal concentration of DMSO. After incubation medium was discarded, and the plates were gently washed three times with 1.5 mL sterile, cold phosphate buffer saline (PBS). Adherent cell were scraped and immediately processed for RNA extraction. All experiments were performed in triplicate (3 independent biological replicates).

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Last updated: 2015-01-27