Multiple roles of RNase Y in Streptococcus pyogenes mRNA metabolism
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ABSTRACT: Streptococcus pyogenes (Group A streptococcus, GAS) is an important human pathogen that causes a variety of infectious diseases and sequelae. Recent studies showed virulence factor expression was controlled at multiple levels, including the post-transcriptional regulation. In this study, we examined the global half-lives of S. pyogenes mRNAs and explored the role RNase Y played in mRNA metabolism with microarray analysis. key word: genetic modification Streptococcus pyogenes NZ131 wild-type cells and ?rny strains were grown in C-medium until late exponential phase. Rifampicin was added to the cell culture and samples were collected before and after rifampicin addition. The transcriptional profile of the whole genome before and after rifampicin addition was examined with microarray. Please note that mRNA decay assay resulted in considerable variations in the datasets. Samples were taken after rifampicin addition and subsequent incubation for different time intervals. During that time no new RNA is produced and the remaining RNA is degraded to various degrees.
Project description:Streptococcus pyogenes (group A Streptococcus, GAS) responds to environmental changes in a manner that results in an adaptive regulation of the transcriptome. Global transcriptional regulators are able to integrate important extracellular and intracellular information and are responsible for modulation of the transcriptional network. The roles of several global transcriptional regulators in adaptation and virulence gene expression have been described. In this study we used microarray to investigate the regulatory roles of CodY and CovRS played in Streptococcus pyogenes. keywords: genetic modification Streptococcus pyogenes NZ131 wild-type cells, ΔcodY, ΔcovRS and ΔcodYcovRS strains were grown in C-medium until mid-exponential phase or early-stationary phase. The transcriptional profile of the whole genome was examined with microarray.
Project description:The speB gene of Streptococcus pyogenes has a long 5' UTR with unknown functions. Preliminary studies showed that the partial mutation of this region led to altered expression patterns of the speB gene and ropB, its upstream gene. In this study, we used microarray to examine the effect of speB 5' UTR mutation on the S. pyogenes transcriptome. The speB ORF and a large portion of the speB 5’ UTR (-1 to -527 from ATG) was replaced with an erythromycin resistant cassette gene (ermAM) to generate a ΔIG mutant. Transcriptome profiles of the wild-type (WT) and ΔIG mutant in the mid-exponential phase and early stationary phase (ST) were examined with microarray analysis.
Project description:Whole genone expression profile comparing wild-type NZ131 to serR deletion mutant, grown in C-medium Mutants and interpretation are described further in the manuscript to be submitted: LaSarre and Federle, 2010. Title: Regulation and Consequence of Serine Catabolism in Streptococcus pyogenes. A two chip study using total RNA recovered from three separate wild-type cultures of Streptococcus pyogenes NZ131 and three separate mutant cultures of Streptococcus pyogenes NZ131 seR-, pooled following RNA extraction
Project description:Expression of the extensive arsenal of virulence factors by Streptococcus pyogenes are controlled by many regulators, of which covR/S is one of the best characterized and can influence ~15% of the genome. Animal models have established that mutants of CovR/S arise spontaneously in vivo resulting in highly invasive organisms. We analyzed a pharyngeal and a blood isolate of S. pyogenes recovered from the same individual 13 days apart. The two isolates varied in many phenotypic properties including speB production, which were reflected in transcriptome analyses. Pulsed field gel electrophoresis, multilocus sequence typing, and partial sequencing of some key genes failed to show any differences except for an 11-base insert in the covS gene in the blood isolate. These results showing that pharyngeal and blood isolates from a single individual which differ by a simple insertion, provide evidence for the model that regulatory gene mutations allow S. pyogenes to invade different niches in the body. A chip study using total RNA recovered from two separate wild-type cultures of group A Streptococcus, Streptococcus pyogenes UH322 and UH328. Each chip measures the expression level of 1865 genes replicated twice from 7 fully sequenced strains of Streptococcus pyogenes (M1_GAS NC_002737; MGAS10394 NC_006086; MGAS315 NC_004070; MGAS5005 NC_007297; MGAS6180 NC_007296; MGAS8232 NC_003485; SSI-1 NC_004606 with fourteen 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Purpose: RNase Y is a major enzyme responsible for mRNA degradation in Streptococcus pyogenes. The goals of this study are to understand whether RNase Y plays a role in operon transcription of S. pyogenes NZ131 by using RNA-seq analysis. Methods: S. pyogenes mRNA profiles of wild type (WT) and RNase Y mutant (∆rny) were generated by deep sequencing, in duplicate, using Illumina Hiseq 2000. The sequence reads were aligned to the S. pyogenes genome using Bowtie2. The aligned files were sorted to BAM format and indexed using Samtools. The read depth of each base was derived from BAM files using BEDtools. Operon organization of S. pyogenes WT and ∆rny strains were predicted based on base reads. Results: A total of 11 to 12 billion reads were obtained from each sample. More than 99% of these reads were mapped to the S. pyogenes genome. Predictions of operon organization using WT and ∆rny samples showed little difference between the two strains. Conclusions: Our result shows that the mutation of RNase Y does not affect the operon organization of S. pyogenes NZ131.
Project description:Streptococcus pyogenes (Group A streptococcus, GAS) is an important human pathogen that causes a variety of infectious diseases and sequelae. Recent studies showed virulence factor expression was controlled at multiple levels, including the post-transcriptional regulation. In this study, we examined the global half-lives of S. pyogenes mRNAs and explored the role RNase Y played in mRNA metabolism with microarray analysis. key word: genetic modification
Project description:Streptococcus pyogenes is a major causative agent of tonsillitis or pharyngitis in children, which can lead to more invasive infections and noninfectious sequellae. S. pyogenes can persist in tonsils, while one-third of children treated with antibiotics continue to shed streptococci and have recurrent infections. Mouse nasal-associated lymphoid tissue (NALT) is functionally analogous to human oropharangeal lymphoid tissues. The innate immune responses of naïve cells from a mucosal site to S. pyogenes is not well described; therefore, we infected C57BL/6 mice intranasally with 108 CFU S. pyogenes. Transcriptional responses by NALT after S. pyogenes infection were analyzed by Affymetrix microarray and quantitative RT-PCR. Wild-type S. pyogenes induces transcription of both type I and IFN-gamma-responsive genes, pro-inflammatory genes, and acute phase response plasma proteins within 24h after infection. Invasion of NALT and the induction of the interferon response were not dependent on expression of anti-phagocytic M1 protein. However, infection with an attenuated, less invasive mutant indicated that a robust innate response by NALT is significantly influenced by intra-NALT bacterial load. Granulocytic populations of NALT, cervical lymph nodes and spleen were discriminated by characteristic surface and intracellular markers. Intranasal infection induces systemic release of neutrophils and a substantial influx of neutrophils into NALT at 24h, which decline by 48h after infection. Macrophages do not significantly increase in S. pyogenes-infected NALT. Intranasal infection of IFN-gamma -/- (GKO) C57BL/6 mice did not lead to systemic dissemination of wild type S. pyogenes, despite reduced expression of IFN-gamma-responsive mRNAs in NALT. Infected GKO mice had an unregulated influx of neutrophils into NALT compared to immunocompetant mice and mice treated with an anti-IFN-gamma antibody more rapidly cleared S. pyogenes from NALT. Thus, IFN-gamma-induced responses have a suppressive influence on early clearance of this pathogen from NALT. Experiment Overall Design: C57BL/6 mice (6-10 weeks old), 4 per group, were infected intranasally with log-phase Streptococcus pyogenes, 2 to 4 x 10^8 CFU per 15 µl of pyrogen-free PBS. Sham-infected mice were administered 15 µl of the same PBS. Mice were infected with wild type strain 90-226 (Cue 1998), a 90-226 strain containing an in-frame deletion of M1 protein (90-226 delta emm1) (Zimmerlein 2005) or an attenuated 90-226 which lacks both M1 and SCPA proteins (90-226att). NALT was collected from mice at 24h after infection and stored in RNAlater until RNA could be purified).
Project description:Whole genome expression profile comparing MGAS315 treated with XIP pheromone versus vehicle-treated cells Interpretations are described further in the manuscript to be submitted: authors Mashburn-Warren, Morrison, and Federle. Title: The Cryptic Competence Pathway in Streptococcus pyogenes is Controlled by a Peptide Pheromone. A two chip study using total RNA recovered from three separate cultures of Streptococcus pyogenes MGAS315, each treated with either XIP pheromone or with vehicle; RNA preparation of cultures receiving same type of treatment were pooled using equivalent amounts of RNA from each culture. RNA pools were fluorescently labeled and hybridized to arrays designed to the S. pyogenes NZ131 genome.
Project description:S. pyogenes strains were compared with the intact covRS form of the globally disseminated M1T1 clone to track transcriptomic changes engendered during the emergence of the M1T1 clone. The mutant covRS form of the M1T1 clone was included as a transcriptomic outlier and to provide a context for the magnitude of transcriptional shifts detected within the isolate set examined. Microarray was performed on RNA extracted from mid-logarithmic phase S. pyogenes grown in Todd-Hewitt with 1% yeast extract in vitro. Experiments were performed using a single color method. Each sample was labelled with Cy3 and hybridized to separate arrays. Each strain was analysed in 3 biological replicates. cDNA hybridized to JCVI PFGRC Streptococcus pyogenes v2 oligo arrays. Only probed representing the core M1 genome were used for analysis.