Unknown,Transcriptomics,Genomics,Proteomics

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Comparative Genome Analysis of Ureaplasma parvum Clinical Isolates

ABSTRACT: Ureaplasma are widespread parasites colonizing the mucosal surface of the human urogenital tract, and it has been suspected as a causative agent of nongonococcal urethritis, pregnancy complications and prenatal infections. Ureaplasma may also cause central nervous system infections and affect the lower respiratory tract of newborn babies. However, Ureaplasma spp. have also been detected in the urogenital tracts of clinically healthy patients, and their role in the development of infections thus remains unclear. Like in other organisms, virulence of Ureaplasma is determined by the presence of virulence factors - adhesions, human IgA protease, phospholipase and urease. However, the existence of interrelationships between the presence of these genes in the Ureaplasma genome and the incidence of diseases in man has not been demonstrated. Difficulties in the elucidation of these interrelationships may arise from significant macro- (gene mutation, chromosomal rearrangements) and micro- (nucleotide polymorphism) genomic heterogeneity. It is possible that the combination of the variable strain-specific genes in Ureaplasma with generally known virulence factors determine the development of pathological processes on the mucosal surface of the human urogenital tract. In our research we used 10 clinical and 1 laboratory strain

ORGANISM(S): Ureaplasma parvum

SUBMITTER: Andrew Klubin

PROVIDER: E-GEOD-5378 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001.

Project description:The GacS/GacA signal transduction system is a central regulator in Pseudomonas spp., including the biological control strain P. fluorescens Pf-5, in which GacS/GacA controls the production of secondary metabolites and exoenzymes that suppress plant pathogens. A whole genome oligonucleotide microarray was developed for Pf-5 and used to assess the global transcriptomic consequences of a gacA mutation in P. fluorescens Pf-5. In cultures at the transition from exponential to stationary growth phase, GacA significantly influenced transcript levels of 632 genes, representing more than 10% of the 6147 annotated genes in the Pf-5 genome. Transcripts of genes involved in the production of hydrogen cyanide, the antibiotic pyoluteorin, and the extracellular protease AprA were at a low level in the gacA mutant, whereas those functioning in siderophore production and other aspects of iron homeostasis were significantly higher in the gacA mutant than in wild-type Pf-5. Notable effects of gacA inactivation were also observed in the transcription of genes encoding components of a type VI secretion system and cytochrome C oxidase subunits. Two novel gene clusters expressed under the control of gacA were identified from transcriptome analysis, and we propose global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp. In this series two conditions have been analyzed. A gacA mutant of Pseudomonas fluorescens Pf-5 was harvested at early (OD 0.5) and late (OD 2.4) time points and compared to wild-type Pf-5 harvested in parallel. For each slide, an experimental RNA sample from a gacA mutant was labeled with Cy3 or Cy5 and was hybridized with a reference RNA sample from wild-type Pf-5 labeled with the other Cy dye. There are six slides per condition. Each condition is represented by three biological replicates. There are two flip-dye replicates for each biological replicate. Each slide contains three replicate spots per gene.

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Comparative Genome Analysis of Ureaplasma parvum Clinical Isolates

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