Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of human hep2 cells infected with Streptococcus pyogenes over time

ABSTRACT: Two biological replicate experiments were performed to estimate the bias of the gene expression pattern of infected and non-infected HEp-2 cells. Microarrays hybridized with RNA from 2 h of non-infected HEp-2 cells were used as reference chips for the comparison with microarrays hybridized with RNA from 2 h and 4 h of eukaryotic cells exposed to wt-bacteria and .fasX-mutant. As a reference for chips hybridized with RNA prepared from 6 h p. i. and 8 h p. i. of both GAS-infected HEp-2 cells we used chips that were hybridized with RNA isolated from non-infected cells 8 h p. i. We also compared the microarray data from 2 h of non-infected HEp-2 cells with those from 8 h of non-infected HEp-2 cells to determine the influence of the extended culture on the non-infected cells. Only such genes which were differentially regulated after infection with wt-bacteria and .fasX-mutant infected cells and not differentially present in unequal amounts between the 2 h and 8 h of controls were included in the subsequent statistical analysis.

ORGANISM(S): Homo sapiens

DISEASE(S): 2h after infection with wildtype Streptococcus pyogenes

SUBMITTER: Dirk Koczan

PROVIDER: E-MEXP-271 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness.

Klenk Michael M Koczan Dirk D Guthke Reinhard R Nakata Masanobu M Thiesen Hans-Jürgen HJ Podbielski Andreas A Kreikemeyer Bernd B

Cellular microbiology 20050901 9

The bacterial human pathogen Streptococcus pyogenes (group A streptococci, GAS) is able to adhere to, internalize into and cross-talk on multiple levels with its host cells. To gain insight into the Fas function in pathogenesis we used Affymetrix human genome DNA-arrays to measure temporal and global transcriptional responses of HEp-2 cells infected with M49 S. pyogenes wild-type bacteria and DeltafasX, an isogenic S. pyogenes two-component-signal-transduction system mutant. A modified stringent ...[more]

PMID: 16098212

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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 naÃ¯ve 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 Âµl of pyrogen-free PBS. Sham-infected mice were administered 15 Âµl 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 complex response of murine macrophages to infection with Streptococcus pyogenes was investigated at the level of gene expression using a high-density oligomer microarray. More than 400 genes were identified as being differentially regulated. Many of the up-regulated genes encoded molecules were involved in immune response and inflammation, transcription, signalling, apoptosis, cell cycle, electron transport and cell adhesion. Of particular interest was the up-regulation of proinflammatory cytokines, typical of the classically activated macrophages (M1 phenotype) such as TNF-?, IL-1 and IL-6, and also the up-regulation of anti-inflammatory mediators such as IL-1ra and IL-10 associated with macrophage alternative activation (M2 phenotype). Furthermore, the gene encoding inducible nitric oxide synthase (iNOS), an enzyme typically implicated in classical activation was not induced in infected macrophages. Instead, the gene encoding arginase, a competitor for the iNOS substrate arginine and involved in the alternative activation pathway was up-regulated in S. pyogenes-infected cells. Thus, the microarray-based gene expression analysis demonstrated that S. pyogenes induced an atypical activation program in macrophages with some but not all features of classically or alternatively activation phenotypes. The microarray data also suggested that the bactericidal activity of macrophages against S. pyogenes is mediated by phagocyte oxydase since p47phox was up-regulated in infected cells. Indeed, the in vivo and in vitro killing of S. pyogenes was markedly diminished in the absence of functional phagocyte (p47phox-/-) but not in the absence of iNOS (iNOS-/-). Understanding how macrophages respond to S. pyogenes at the molecular level may facilitate the development of new therapeutic paradigms. Experiment Overall Design: Total RNA was isolated from highly purified F4/80+ cells obtained from the peritoneal cavity of C3H/HeN infected and uninfected control mice and hybridized to an Affymetrix GeneChip MOE430A.

Dataset Information

Transcription profiling of human hep2 cells infected with Streptococcus pyogenes over time

Publications

Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness.

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