Project description:The polyamine biosynthesis gene, speE, in Streptococcus pneumoniae TIGR4 is necessary for survival in murine models of pneumococcal pneumonia. To date, there is no description of polyamine biosynthesis dependent pneumococcal gene expression. In this study, we compared gene expression between the wild-type and biosynthesis deficient (speE) TIGR4 by RNA-Seq analysis.
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).
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 polyamine transport operon in Streptococcus pneumoniae TIGR4 is necessary for survival in murine models of pneumococcal pneumonia. To date, there is no description of polyamine transport dependent pneumococcal gene expression. In this study, we compared gene expression between the wild-type and transport deficient (potABCD) TIGR4 by RNA-Seq analysis.
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:Streptococcus pneumoniae (S.p.) is the most common causative agent of community-acquired pneumonia worldwide. A key pathogenic mechanism that exacerbates severity of disease is the disruption of the alveolo-capillary barrier. However, the specific virulence mechanisms responsible for this in the human lung are not yet fully understood. RNA sequencing of Streptococcus pneumoniae transcriptome under infection media conditions, but without the presence of lung tissue, representing anon-host-infection scenario FCS+/- was analyzed,. RNA isolation was performed using an acidic phenol-chloroform extraction protocol (Wetzstein et al., 1992). After DNase-I treatment (Zymo Research, Germany), the RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). For RNA-seq transcriptomics, Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, United States) was applied to prepare the cDNA libraries. The cDNAs were sequenced paired end on an Illumina HiSeq 1500 (San Diego, CA, United States) using 70 and 75 bp read length and a minimum sequencing depth of 10 million reads per library.