Activation by CcpA-FDP of two key determinants of pneumococcal virulence, capsule production and choline utilization.
Ontology highlight
ABSTRACT: The kinetic behaviour of S. pneumoniae during vaccine production was studied using a dynamic systemic approach. Quantification of key intracellular glycolytic metabolites coupled to global transcriptomic analysis led to an improved knowledge of pneumococcal physiology. In controlled growth conditions, a direct correlation between the accumulation of key glycolytic intermediates and the expression of capsular polysaccharide genes (cps operon encoding the main pneumococcal antigen) was established. Interestingly, the same correlation was confirmed for the genes involved in the assimilation and the modification of choline, an indispensable nutritional requirement for both growth and virulence of S. pneumoniae. Such a correlation suggests a direct or indirect control of the expression of these genes by the global transcriptional regulator CcpA (catabolite control protein A) since putative cre sites upstream of the promoter were identified, even though the exact nature of this regulation remains to be confirmed. Preliminary results indicate that a ccpA mutation provokes a significant decrease in the expression of these genes. The global transcriptomic analysis coupled with motif research has revealed an extended CcpA regulon in S. pneumoniae including genes involved in diverse metabolic functions. Keywords: Capsular polysaccharide industrial production One condition experiment examining the transcriptome between two growth phases of the same serotype (slide 1 to slide 4). Thus, cross analysis between the two serotypes during exponential growth was performed in order to complete the analysis
Project description:Several genes involved in nitrogen metabolism are known to contribute to the virulence of pathogenic bacteria. Here, we studied the function of the nitrogen regulatory protein GlnR in the Gram-positive human pathogen Streptococcus pneumoniae. We demonstrate that GlnR mediates transcriptional repression of genes involved in glutamine synthesis and uptake (glnA, glnPQ), glutamate synthesis (gdhA), and the gene encoding the pentose phosphate pathway enzyme Zwf, which forms an operon with glnPQ. Moreover, the expression of gdhA is also repressed by the pleiotropic regulator CodY. The GlnR-dependent regulation occurs through a conserved operator sequence and is responsive to the concentration of glutamate, glutamine and ammonium in the growth medium. By means of in vitro binding studies and transcriptional analyses we show that the regulatory function of GlnR is dependent on GlnA. Mutants of glnA and glnP displayed significantly reduced adhesion to Detroit 562 human pharyngeal epithelial cells, suggesting a role for these genes in the colonization of the host by S. pneumoniae. Thus, our results provide a thorough insight into the regulation of glutamine and glutamate metabolism of S. pneumoniae as mediated by both GlnR and GlnA. Each amplicon was spotted twice (technical replicates) on the DNA microarray. These replicates are indicated in the platforms and DNA microarray data by the addition of _rep1, _rep2, etc. The RNA for wild-type and mutant strains was isolated in 3 independent biological replicates which were hybridized in (partly dye-swap) to the 3 slides for glnA and 3 slides for glnR.
Project description:Oxidative stress, resulting from an imbalance in the accumulation and removal of reactive oxygen species such as hydrogen peroxide (H2O2), is a challenge faced by all aerobic organisms. In plants, exposure to various abiotic and biotic stresses results in accumulation of H2O2 and oxidative stress. Increasing evidence indicates that H2O2 functions as a stress signal in plants, mediating adaptive responses to various stresses. To analyze cellular responses to H2O2, we have undertaken a large-scale analysis of the Arabidopsis transcriptome during oxidative stress. Using cDNA microarray technology, we identified 175 non-redundant expressed sequence tags that are regulated by H2O2. Of these, 113 are induced and 62 are repressed by H2O2. A substantial proportion of these expressed sequence tags have predicted functions in cell rescue and defense processes. RNA-blot analyses of selected genes were used to verify the microarray data and extend them to demonstrate that other stresses such as wilting, UV irradiation, and elicitor challenge also induce the expression of many of these genes, both independently of, and, in some cases, via H2O2. replicate_design
Project description:The bacteriostatic and bactericidal effects and the corresponding expression profiles of Mycobacterium tuberculosis to representative oxidative and nitrosative stresses were investigated by growth and survival studies and whole genome expression analysis. The response of M. tuberculosis to a range of hydrogen peroxide (H2O2) concentrations tended to fall into three distinct categories: (1) low level exposure resulted in induction of few H2O2 sensitive genes, (2) intermediate exposure resulted in massive transcriptional changes without an effect on growth or survival, and (3) high exposure resulted in a muted transcriptional response and eventual death. Nitric oxide (NO) exposure initiated much of the same transcriptional response as H2O2. However, unlike H2O2 exposure, NO exposure affected a dose-dependent bacteriostatic activity without killing and induction of dormancy-related genes. Included in the shared response to H2O2 and NO was the induction of genes encoding oxidative stress detoxification and iron-sulfur cluster repair functions. Expression of several key oxidative stress defense genes was constitutive, or increased moderately from an already elevated level, suggesting bacilli that are continually primed for oxidative stress defense. Deletion of the known oxidative stress responsive regulator, FurA, resulted in the constitutive expression of furA, katG, and Rv1907c; while other genes do not appear to be solely controlled by FurA. In contrast to Escherichia coli, M. tuberculosis appears highly resistant to DNA damage-dependent killing caused by low mill molar levels of H2O2. Furthermore, instead of limiting access to iron to prevent hydroxyl radical formation from H2O2 and thus DNA damage, M. tuberculosis induced iron uptake genes in response to H2O2 and NO. Set of arrays that are part of repeated experiments Compound Based Treatment: H2O2 or DETA/NO treatment
Project description:Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. User Defined
Project description:There are about 800 genes in Saccharomyces cerevisiae whose transcription is cell-cycle regulated. Some of these form clusters of co-regulated genes. The 'CLB2' cluster contains 33 genes whose transcription peaks early in mitosis, including CLB1, CLB2, SWI5, ACE2, CDC5, CDC20 and other genes important for mitosis. Here we find that the genes in this cluster lose their cell cycle regulation in a mutant that lacks two forkhead transcription factors, Fkh1 and Fkh2. Fkh2 protein is associated with the promoters of CLB2, SWI5 and other genes of the cluster. These results indicate that Fkh proteins are transcription factors for the CLB2 cluster. The fkh1 fkh2 mutant also displays aberrant regulation of the 'SIC1' cluster, whose member genes are expressed in the M-G1 interval and are involved in mitotic exit. This aberrant regulation may be due to aberrant expression of the transcription factors Swi5 and Ace2, which are members of the CLB2 cluster and controllers of the SIC1 cluster. Thus, a cascade of transcription factors operates late in the cell cycle. Finally, the fkh1 fkh2 mutant displays a constitutive pseudohyphal morphology, indicating that Fkh1 and Fkh2 may help control the switch to this mode of growth. Groups of assays that are related as part of a time series. Computed
Project description:The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Computed
Project description:While several groups, including our own, have examined variability of M. tuberculosis at the DNA level, this is the first systematic survey of variability in mRNA expression among clinical isolates of M. tuberculosis. Genes whose expression varies among isolates when assayed under a single growth condition may make poor drug targets and vaccine antigens and may affect molecular diagnostics, so they can be used to narrow down lists of candidate molecules. Because the measurement of gene expression is extremely sensitive to environmental conditions, comparison of gene expression is labour intensive. In this study, we surveyed 12 strains. These strains are a subset of those for which we have already published genomic deletion information (Kato-Maeda et al., 2001). In order to ensure maximum reproducibility of the experiments and avoid complications caused by differences in growth conditions, we measured gene expression under well-controlled in vitro conditions. Our aims were to provide an overview of gene expression variability among clinical isolates under a single growth condition and to test whether gene functional classes are related to variability in expression. Set of arrays that are part of repeated experiments Keywords: Biological Replicate Biological Replicate Computed
Project description:Cells infected with the intracellular protozoan parasite Toxoplasma gondii undergo upregulation of pro-inflammatory cytokines, organelle redistribution, and protection from apoptosis. To examine the molecular basis of these and other changes, gene expression profiles of human foreskin fibroblasts infected with Toxoplasma were studied using human cDNA microarrays consisting of ~22,000 known genes and uncharacterized ESTs. Early during infection (1-2 h), <1% of all genes show a significant change in the abundance of their transcripts. Of the 63 known genes in this group, 27 encode proteins associated with the immune response. These genes are also upregulated by secreted, soluble factors from extracellular parasites indicating that the early response does not require parasite invasion. Later during infection, genes involved in numerous host cell processes, including glucose and mevalonate metabolism, are modulated. Many of these late genes are dependent on the direct presence of the parasite; i.e. secreted products from either the parasite or infected cells are insufficient to induce these changes. These results reveal several previously unknown effects on the host cell and lay the foundation for detailed analysis of their role in the host-pathogen interaction. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
Project description:Host cell infection by the intracellular pathogen, Trypanosoma cruzi, involves activation of signaling pathways, cytoskeletal reorganization, and targeted recruitment of host cell lysosomes. To determine the consequences of T. cruzi invasion on host cell gene expression, high density microarrays consisting of approximately 27,000 human cDNAs were hybridized with fluorescent probes generated from T. cruzi-infected human fibroblasts (HFF) at early time points following infection (2-24 h). Surprisingly, no genes were induced > or =2-fold in HFF between 2 and 6 h post-infection (hpi) in repeated experiments while immediate repression of six host cell transcripts was observed. A significant increase in transcript abundance for 106 host cell genes was observed at 24 hpi. Among the most highly induced is a set of interferon-stimulated genes, indicative of a type I interferon (IFN) response to T. cruzi. In support of this, T. cruzi-infected fibroblasts begin to secrete IFNbeta at 18 hpi following the induction of IFNbeta transcripts. As compared with global transcriptional responses evoked by other intracellular pathogens, T. cruzi is a stealth parasite that elicits few changes in host cell transcription during the initiation of infection. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
Project description:The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2-dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2. To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2-HIS3 fusion in the absence of H2O2. Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase. Northern blot and whole-genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2-inducible genes is elevated in Deltatrr1 mutants in the absence of external stress. In Deltatrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein-folding chaperones, are hyperinduced. However, despite the elevated expression of genes encoding antioxidant enzymes, Deltatrr1 mutants are extremely sensitive to H2O2. The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress-induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity. Groups of assays that are related as part of a time series. Using regression correlation