Project description:Otitis media, pneumonia, sinusitis and as well as severe diseases such as meningitis and bacteraemia are related to biofilm-like diseases, in which Streptococcus pneumoniae demonstrated differential and tissue specific gene expressions. In this study, we reported the differential gene expression profile of early in vitro biofilm and planktonic cell in c-DNA microarray analysis. The microarray analysis was performed on total RNA extracted from biofilms grown in 24-well microtiter plate and mid-log grown planktonic cells. To validate the results of microarray, real-time RT-PCR was performed on 13 differentially expressed genes and one constitutively expressed gene from six different functional groups. cDNA-microarray analyses indicated 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Among total differentially expressed gene, almost 50% were hypothetical genes. Of the 46 protein coding genes, 34 showed up-regulation and 16 showed down-regulation in biofilm. The functional annotation showed that many functional categories were differentially regulated in biofilm and planktonic cells, such as genes involve in purine, pyrimidine nucleotide metabolism, RNA/DNA metabolism, amino acid transport and metabolism, translation, transporter protein, carbohydrate transport and metabolism, cell wall biosynthesis, isoprenoid biosynthesis, transcription regulator and cellular process.
Project description:Otitis media, pneumonia, sinusitis and as well as severe diseases such as meningitis and bacteraemia are related to biofilm-like diseases, in which Streptococcus pneumoniae demonstrated differential and tissue specific gene expressions. In this study, we reported the differential gene expression profile of early in vitro biofilm and planktonic cell in c-DNA microarray analysis. The microarray analysis was performed on total RNA extracted from biofilms grown in 24-well microtiter plate and mid-log grown planktonic cells. To validate the results of microarray, real-time RT-PCR was performed on 13 differentially expressed genes and one constitutively expressed gene from six different functional groups. cDNA-microarray analyses indicated 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Among total differentially expressed gene, almost 50% were hypothetical genes. Of the 46 protein coding genes, 34 showed up-regulation and 16 showed down-regulation in biofilm. The functional annotation showed that many functional categories were differentially regulated in biofilm and planktonic cells, such as genes involve in purine, pyrimidine nucleotide metabolism, RNA/DNA metabolism, amino acid transport and metabolism, translation, transporter protein, carbohydrate transport and metabolism, cell wall biosynthesis, isoprenoid biosynthesis, transcription regulator and cellular process. Streptococcus pneumoniae R6 strain used in this is an unencapsulated and avirulent strain derived from encapsulated serotype 2 pathogenic strain D39. 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 supplied with 1% glucose, inoculated 1.5 mL in 24-well microtiter plate and, incubated for 15 hours at 37M-BM-0C in 5% CO2. 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. For planktonic cells RNA extraction, five ml of mid-logarithmic phase cell suspension was pelleted by centrifugation and wash three times with sterile PBS and immediately processed for RNA extraction. All experiments were performed in triplicate (3 independent biological replicates)
Project description:Comparative analyses of protein expression between log-phase planktonic and 1-day and 7-day biofilm cultures from Streptococcus pneumoniae representing nascent and late phase biofilm growth were performed using the proteomic approach iTRAQ
Project description:Carolacton is a novel biofilm inhibitor that kills biofilm cells of Streptococcus mutans in nanomolar concentrations. Interestingly, Carolacton also inhibits growth of the clinically relevant and human pathogenic bacterium Streptococcus pneumoniae TIGR4. The cellular target of Carolacton is still unknown. Here, we adressed the differential transcription of cellular RNAs when S. pneumoniae TIGR4 was grown in the presence of Carolacton. This was done to identify transcriptional regulatory networks that are directly affected by treatment of the pneumococcus with Carolacton. In order to gain insights into the primary transcriptional response, early time-points were chosen for sampling, which should not reflect secondary responses (e.g. due to differences in growth phase, drop in pH etc.). To achieve a thorough overview over all affected cellular RNA species, such as mRNAs, small regulatory RNAs and tRNAs, and not to lose small transcripts during library preparation, RNAs were separated according to size and used to construct two separate libraries for sequencing.
Project description:The most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Separate domains were identified depending of the transcription of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show here that these distinct domains have different expression and conservation tendencies. Microarray fluorescence units under non-relaxation conditions, taken as a measure of gene transcription level, were significantly lower in F genes than in the other domains in the same range of AT content. Transcription level categorization of the domains was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences evidenced conservation of gene composition in the U and D domains and extensive gene interchange in F domains. We tested domain organization by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology works as a general transcriptional regulator, superimposed to other kind of more specific regulatory mechanisms.
Project 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.
Project description:This SuperSeries is composed of the following subset Series: GSE31815: ccpA mutant compared to D39 wild-type in Streptococcus pneumoniae in CDM + Glucose at MID-log growth phase GSE31816: ccpA mutant compared to D39 wild-type in Streptococcus pneumoniae in CDM + GLucose at transition-phase of growth (TS) GSE31817: ccpA mutant compared to D39 wild-type in Streptococcus pneumoniae in CDM + Galactose at MID-log growth phase GSE31818: ccpA mutant compared to D39 wild-type in Streptococcus pneumoniae in CDM + galactose at transition-phase of growth (TS) Refer to individual Series
Project description:To investigate the gene transcription profiles of Streptococcus suis in the planktonic state and biofilm state, we provided samples cultured for 8 h in the planktonic state and samples cultured for 24 h in vitro biofilm. We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 different samples at two time points.