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:Transcriptomic analysis of Streptococcus pneumoniae TIGR4 wildtype and it's isogenic ccpA mutant, grown in chemically defined media supplemented with physiological levels of carbohydrates to mimic the human nasopharynx and bloodstream. The goal is to examine how anatomical site-specific carbohydrate availability impacts S. pneumoniae physiology and virulence.
Project description:Invasive pneumococcal disease is preceded by asymptomatic colonization of the human nasopharynx by Streptococcus pneumoniae. Progression from colonization to invasion is a watershed in the host-pathogen interaction, and exposes the pneumococcus to markedly different microenvironments. This in turn, requires alterations in gene expression profile to adapt to the new niche. One apparent adaptive mechanism is reversible phase variation between “transparent” and “opaque” colony opacity phenotypes. Transparent phase variants colonize the nasopharynx more efficiently than opaque variants of the same strain, while opaque variants exhibit higher systemic virulence. Previous studies have reported quantitative differences in surface components such as the capsule, teichoic acid and certain surface proteins between the two phenotypes, but the underlying regulatory mechanism is not understood. In the present study, we found no differences in expression of key surface proteins between opaque and transparent variants of S. pneumoniae strain D39, but opaque cells produced five-fold more capsular polysaccharide. Subsequent microarray and real-time RT-PCR analysis showed no differences in capsule gene expression, but several genes involved in uridine monophosphate (UMP) biosynthesis were up-regulated in the opaque phenotype. This correlated with significant increases in the intracellular concentrations of both UMP and UDP-glucose, which are essential precursors for capsule biosynthesis. Our data suggest a novel mechanism for pneumococcal capsule regulation, in which rate-limiting precursor pathways are modulated rather than the capsule biosynthetic genes themselves. Keywords: Phase variants
Project description:Microarray analysis of Streptococcus pneumoniae TIGR4 transcriptome in response to manganese as the transcriptome changes in response to intracellular manganese accumulation via a mutation in sp1552/mntE a manganese efflux protein. Investigating role of manganese efflux and accumulation in S. pneumoniae: 3 TIGR4 in ThyB vs. TIGR4 in Mn and 3 TIGR4 in Mn vs mntE1 in Mn replicate 3
Project description:TIGR4 and R6 bacterial strains of Streptococcus pneumoniae treated and not treated with the iron chelator deferoxamine mesylate (DFO)
Project description:Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and a leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by post-transcriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs have been further implicated in regulating pneumococcal pathogenesis. However, there is little overlap in the sRNAs identified among these studies, which indicates that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there are likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery's virulent S. pneumoniae strain D39 using two independent RNA-seq based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program towards identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4 including ones previously implicated in virulence are not present in strain D39 genome suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 67 new sRNA candidates in strain D39, 28 out of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112.
Project description:Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and the leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by post-transcriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs have been further implicated in regulating pneumococcal pathogenesis. However, there was little overlap in the sRNAs identified among these studies, which indicated that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there were likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery's virulent S. pneumoniae strain D39 using two independent RNA-seq based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program towards identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4 including ones previously implicated in virulence were not present in strain D39 genome suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 67 new sRNA candidates in strain D39, 28 out of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112.
Project description:Microarray analysis of Streptococcus pneumoniae TIGR4 transcriptome in response to manganese as the transcriptome changes in response to intracellular manganese accumulation via a mutation in sp1552/mntE a manganese efflux protein.
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:Transcriptional responses of Streptococcus pneumoniae strain TIGR4 to human lung epithelial cells (A549) were investigated at the early stages of interactions (0.5 h and 1 h).