Project description:Streptococcus pneumoniae (pneumococcus) is a leading human pathogen that can cause serious localized and invasive diseases. Pneumococci can undergo a spontaneous and reversible phase variation that is reflected in colony opacity, which allows the population to adapt to different host environments. Generally, transparent variants are adapted for nasopharyngeal colonization whereas opaque variants are associated with invasive disease. In recent work, colony phase variation was shown to occur by means of recombination events to generate multiple alleles of the hsdS targeting domain of a DNA methylase complex, which mediates epigenetic changes in gene expression. A panel of isogenic strains were created in the well-studied S. pneumoniae TIGR4 background that are “locked” in the transparent (n=4) or opaque (n=2) colony phenotype. The strains had significant differences in colony size which were stable over multiple passages in vitro and in vivo. While there were no significant differences in adherence for the phase-locked mutant strains to immortalized epithelial cells, biofilm formation and viability was reduced for opaque variants in static assays. Nasopharyngeal colonization was stable for all strains, but the mortality rate differed between them. Transcript profiling by RNA-seq analyses revealed that expression of certain virulence factors were increased in a phase-specific manner. As epigenetic regulation of phase variation (often referred to as phasevarion) is emerging as a common theme for mucosal pathogens, these studies serve as a model for future studies of host-pathogen interactions.
Project description:The objective of the study is to obtain a high resolution transcription map of S. pneumoniae genome and make it available through a genome browser. The study also help to identify gene expression pattern, presence of small RNAs, Antisense expression and operon structure.
Project description:Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease
Project description:The objective of the study is to obtain a high resolution transcription map of S. pneumoniae genome and make it available through a genome browser. The study also help to identify gene expression pattern, presence of small RNAs, Antisense expression and operon structure. S. pneumoniae strain TIGR4 (ref) was grown in THYmedium, Todd-Hewitt broth supplemented with 0.5% yeast extract. Cells were harvested from duplicate cultures from mid-log phase (OD600 nm, 0.4â0.6) of growth by centrifugation. The harvested pellets were washed twice in sterile PBS (pH 7.0) and stored at -80°C. RNA was purified from frozen bacterial pellets using Qiagen RNeasy kit by following the manufacturerâs protocol. Isolated RNA was treated with DNase to remove genomic DNA contamination, and purity was checked by performing a one-step RT-PCR using primers specific for 16S rRNA in presence or absence of reverse transcriptase. RNA concentration and quality were determined by using Agilent Bioanalyzer. Purified RNA was stored in nuclease free water at -80°C. One microgram of total RNA was used by Nimblegen systems for labeling and hybridization. 2 biological replicates (single colored) were used in the analysis. 20,000 random probes were designed on the chip as a negative controls to measure non-specific hybridization. 35 genes identified through proteomics of same sample were used as positive controls.
Project description:Purpose: We recently reported that isogenic deletion of lysine decarboxylase (ΔcadA/SP_0916), an enzyme that catalyzes the biosynthesis of polyamine cadaverine in Streptococcus pneumoniae TIGR4 results in loss of capsular polysaccharide (CPS), which constitutes a novel mechanism of regulation of CPS. Here, we conducted RNA-Seq to elucidate molecular mechanisms of CPS regulation in polyamine synthesis impaired pneumococci. Result: Significantly differentially expressed genes in ΔcadA represent pneumococcal pathways involved in the biosynthesis of precursors for CPS and peptidoglycan. Conclusion: We establish a possible link and interchange between two cellular processes such as high energy demanding capsule production and oxidative stress responses in polyamine synthesis impaired pneumococci (ΔcadA).
