Unknown,Transcriptomics,Genomics,Proteomics

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Comparative genomics and transcriptomics of Propionibacterium acnes

ABSTRACT: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal, but is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with health. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a sequence type (ST) 18 strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits, differ between phylotypes. Comparative transcriptome analysis revealed that 225 genes of strain KPA171202 (type I-2, ST34) were differentially transcribed in strain 266 during exponential growth. 47% of these genes belong to the strain-specific gene content of strain KPA171202, indicating that strain-specific functions are utilized. Next, we studied differential expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by up-regulation of genes involved in the stress response and amino acid metabolism. Taken together, our data highlight the genomic basis for strain diversity and identify, for the first time, the transcribed part of the genome, underling the important role active growth plays in the inflammatory activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by variable genome content but also, and to a greater degree, by variable transcriptomes. Microarray experiments were performed as dual-color hybridizations. In order to compensate specific effects of the dyes and to ensure statistically relevant data analysis, a color-swap dye-reversal was performed. Two different labeling methods were applied. RNA labeling was performed with the two color Quick Amp Labeling Kit (Agilent Technologies) using FullSpectrum MultiStart Primer for T7 IVT RNA Amplification (BioCat GmbH, Heidelberg, Germany) as random T7 labeling. Alternatively, the total RNA samples were amplified with the TransPlex Whole Transcriptome Amplification Kit (Sigma-Aldrich, Munich, Germany) and labeled with BioPrime Plus Array CGH Indirect Genomic Labeling System (Invitrogen, Karlsruhe, Germany) as WTA BioPrime indirect. Both methods were compared and combined for final data analysis.

ORGANISM(S): Propionibacterium acnes

SUBMITTER: Hans-Joachim Mollenkopf

PROVIDER: E-GEOD-26738 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Proprionibacterium acnes is a Gram positive bacterium found ubiquitously on human skin, where it is typically considered to assume a commensal relationship with its host. However, it is also closely associated with the skin condition acne vulgaris. More controversially, it has a postulated involvement in infections of implanted prosthetic devices and has been isolated from malignant prostate tissues. The role of P. acnes in these pathologies remains undetermined, although both bacterial and host factors are implicated. By microarray analysis, we have identified fundamental differences in the global transcriptional profiles of keratinocyte and prostate cells to P. acnes infection. Notably, P. acnes infection of the keratinocyte cell line, HaCaT, elicited a robust, but acute inflammatory response. By contrast, the inflammatory response of the prostate cell line, RWPE1, was delayed and persisted for longer times. Immunofluorescence and electron microscopy revealed higher numbers of internalized P. acnes bacteria in RWPE1 cells, which could still be detected intracellularly three weeks post infection. This contrasted with HaCaT cells, which P. acnes largely failed to invade. Moreover, P. acnes induced a delayed, sustained activation of NFM-NM-:B in RWPE1 cells, which was absent in HaCaT cells. Further characterization of the host-cell response to infection revealed that the intermediate filament protein, vimentin, was a key determinant of P. acnes invasion, persistence and inflammatory profile in RWPE1 cells. siRNA mediated knock down of vimentin in RWPE1 cells attenuated bacterial invasion and the inflammatory response to infection; similarly, overexpression of vimentin in HaCaT cells increased bacterial invasion. We conclude that vimentin-dependent host-tissue tropism, in part, determines P. acnes invasion and inflammatory capacity. This could contribute to P. acnes pathology at non-skin infection sites. Microarray experiments were performed as dual-color hybridizations. In order to compensate specific effects of the dyes and to ensure statistically relevant data analysis, a color-swap dye-reversal was performed.

Project description:MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly up-regulate NF-κB associated microRNAs; strikingly, these regulations which are induced by the bacterial lipopolysaccharides (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxintolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens. The murine macrophage cell-line RAW 264.7 was exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. We compared mouse mRNA abundances between cells infected with wild-type ("24WT") or SPI1/2 knockout Salmonella ("24SPI1/2") and non-infected, mock-treated controls ("24C"). Both strains mounted a similar inflammatory response as judged from the abundance of pro-inflammatory genes compared to the controls. These data indicate, that macrophages can mount a full inflammatory response to extracellular bacteria, that is not further enhanced or attenuated by the sensing of intracellular bacteria. Murine RAW 264.7 cells were rendered endotoxin-tolerant by pre-exposure to heat-killed Salmonella typhimurium SL1344 ("HKS") at an MOI of 10 for 20 h. 5 days upon removal of the HKS stimulus cells were exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. The wild-type Salmonella strain invades and replicates in endotoxin-tolerant RAW 264.7 cells, though the intracellular replication rate is significantly reduced (by roughly 40 %) compared to naive RAW 264.7 cells. We compared mouse mRNA abundances between cells infected with wild-type ("wt") and non-infected, mock-treated controls ("Mock") or between wild-type ("wt") and SPI1/2-knockout ("SPI1/2") infected cells. As apparent from the Microarray data the wild-type Salmonella strain induces a typical inflamatory response in endotoxin-tolerant RAW 264.7 cells (as judged from the fold-induction of NFkB-inducible genes). As expected however, mRNAs corresponding to known inflammatory genes are significantly less abundant in response to the non-invasive SPI1/2 knockout strain, compared to wild-type infection. These data indicate, that endotoxin-tolerance renders macrophages largely insensitive to extracellular, but not to intracellular bacteria. ***This submission represents the mRNA component of the study

Project description:Despite extensive studies, the fundamental mechanisms responsible for the development and progression of heart failure have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species (ROS) play a major pathological role. Quercetin, a major flavonoid present in the human diet, has been studied widely and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. This study was therefore designed to examine the gene expression profiling of cultured rat primary cardiomyocytes supplemented with quercetin using DNA-microarrays and relating these data to functional effects. Experiment Overall Design: Primary heart cell cultures were obtained by isolation of cardiomyocytes from the ventricles of 2â4 day old Wistar rats and grown until complete confluence. In some dishes, 30 Î¼M quercetin was added to the culture medium for 6, 12 or 24 h. Total RNA was extracted using Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA), following the manufacturerâs protocol. The yield and purity of the RNA were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Rockland, Del, USA). The 28s and 18s ribosomal bands were checked using Agilent 2100 bioanalyzer with an RNA 6000 Nano LabChip Kit. For each sample, mRNA was amplified starting from 1.5 Î¼g of totRNA by Amino Allyl MessageAmp I aRNA Kit (Ambion Inc.) to obtain amino allyl antisense RNA (aaRNA). Labeling of aaRNA was performed using NHS ester Cy3 or Cy5 dies (Amersham Biosciences, Piscataway, NJ - USA). One hybridization to Agilent 22K-gene arrays (Rat oligo array G4130A) was performed to compare quercetin supplemented cells versus unsupplemented ones. Each analysis was replicated swapping the labeling with the two cyanine dyes.

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Comparative genomics and transcriptomics of Propionibacterium acnes

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