Project description:This transcriptional analysis is a follow up to a population genomic investigation of 3615 Streptococcus pyogenes serotype M1 strains whch are responsible for an epidemic of human invasive infections (www.pnas.org/cgi/doi/10.1073/pnas.1403138111), The goal was to assess gene expression differences between predecessor pre-epidemic M1 strains and their descendent epidemic M1 strains to gain insights into the underlying genetic basis for the shift in the frequency and severity of human infections caused by these pathogenic bacteria
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. Keywords: disease state analysis
Project description:This transcriptional analysis is a follow up to a population genomic investigation of 3615 Streptococcus pyogenes serotype M1 strains whch are responsible for an epidemic of human invasive infections (www.pnas.org/cgi/doi/10.1073/pnas.1403138111), The goal was to assess gene expression differences between predecessor pre-epidemic M1 strains and their descendent epidemic M1 strains to gain insights into the underlying genetic basis for the shift in the frequency and severity of human infections caused by these pathogenic bacteria The transcriptomes of 7 GAS M1 strains, 4 pre-epidemic and 3 epidemic, were compared at two phases of growth, mid-exponential and early-stationary, using 3 biologial replicates, to identify genes differentially expressed between the pre-epidemic and epidemic isolates with the goal of to gaining insight into the underlying genetic basis for the evolutionary emergence, increased frequency and severity of the epidemic strains relative to the pre-epidemic strains
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:The human pathogen Streptococcus pyogenes, or group A streptococcus, is responsible for mild infections to life-threatening diseases. We previously have performed the transcription start site profiling of a Streptococcus pyogenes emm1 strain, strain S119, an invasive strain isolated from a blood culture. Here, we perform strand-specific RNA-seq experiments to complete this characterization and analyze the global coverage and the differential expression in growth medium complemented or not with 15 mM MgCl2. In addition we compare these results to those obtained with a related strain, strain S126, corresponding to a colonization sample, that differs from S119 by only one mutation in the two-component regulator of virulence CovRS.
Project description:The objective of this study was to investigate which genes are important for Streptococcus pyogenes during intracellular survival in human macrophages. Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is yet unknown. To address this, gene expression profiles of S. pyogenes within human macrophages were determined and compared to those of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection and within 2 h post infection, the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key upregulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). We observed that an isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts as compared to wild-type bacteria following infection of macrophages. Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurrent with an upreguation of the well-studied covR/S two component regulator. In reinfection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems.
Project description:The objective of this study was to investigate which genes are important for Streptococcus pyogenes during intracellular survival in human macrophages. Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is yet unknown. To address this, gene expression profiles of S. pyogenes within human macrophages were determined and compared to those of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection and within 2 h post infection, the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key upregulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). We observed that an isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts as compared to wild-type bacteria following infection of macrophages. Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurrent with an upreguation of the well-studied covR/S two component regulator. In reinfection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems. Five samples with three biological replicates are analysed. Each open reading frame in triplicate (three technical replicas per sample). Resulting in 6-9 data points per gene per condition. The extracellular bacteria are control samples and the internal control is the house-keeping gene gyrase.