Project description:We aim to compare global transcriptomic analysis of wt and delta-nrdRmutant in Pseudomonas aeruginosa PAO1 during aerobic and anaerobic conditions.
Project description:The goals of this experiment was to obtain a transcriptional profile of EHEC 86-24 upon deletion of fusK or fusR genes. The samples were grown in DMEM until late logaritmic phase then RNA was extracted using the TriZol method.
Project description:Escherichia coli O157:H7 is a food-borne pathogen that causes bloody diarrhea and hemolytic uremic syndrome. Hfq is an sRNA chaperone protein that is involved in post-transcriptional regulation of virulence genes in pathogenic bacteria. In EHEC strain EDL933, Hfq acts a negative regulator of the locus of enterocyte effacement (LEE) that encodes most of the proteins involved in type three secretion and attaching and effacing lesions. We deleted hfq in E. coli O157:H7 strain 86-24 and compared global transcription profiles of the hfq mutant to the wild type strain in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler as well as genes encoded in LEE2-5 that encode for type three secretion and AE lesion formation. Decreased expression of the LEE genes in the hfq mutant occurred at mid-, late, and stationary growth phases in both LB and DMEM media as detected by qRT-PCR. We also confirmed decreased regulation of the LEE genes by examining secreted proteins and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC involved in inter-kingdom signaling and virulence gene regulation in EHEC as well as an increase in stx2AB expression that encodes for the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a different regulatory role in EHEC 86-24 from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE. Comparison of transcriptional regulation of the WT 86-24 isolate and the hfq mutant for the identification of regulated targets that were followed up by functional analysis.
Project description:Atypical EPEC (aEPEC) strains are part of group of pathogens capable of forming the Attaching and Effacing (A/E) lesion. This lesion is characterized by intimate adherence of bacteria to enterocytes, and microvilli destruction. The genes responsible to cause that lesion are located in a pathogenicity island called Locus of Enterocyte Effacement (LEE). Transcription of LEE genes is subjected to various levels of regulation, including quorum sensing through autoinducer 3 (AI-3) system. AI-3 is an aromatic compound with similar characteristics to the epinephrine and norepinephrine hormones. This similarity allows bacteria to use these hormones and AI-3 to perform cell M-bM-^@M-^S to M-bM-^@M-^S cell signaling processes and bacteria - host communication processes in order to modulate its virulence. AI-3, epinephrine and norepinephrine are detected by a sensor kinase named quorum sensing E.coli regulator (QseC). In order to investigate the role of QseC and epinephrine in atypical EPEC O55:H7 virulence, we constructed a QseC mutant of this strain and performed transcription and phenotypic analyses in the presence or absence of epinephrine. We have reported here, for the first time, the quorum sensing QseC regulation of virulence genes in atypical EPEC. Our results shown that QseC is a global regulator of gene expression in aEPEC and positively regulates flagellar genes, LEE and non-LEE encoded factors. We also have shown that the presence of epinephrine could be sensed by other receptor that acts as negative regulator of LEE4 and LEE5 genes. Comparison of transcriptional regulation of enteropathogenic E. coli serotype O55:H7 wild type and the qseC mutant in the absence or presence of epinephrine signal to identify the regulated targets
Project description:Enterohemorrhagic E. coli (EHEC) colonizes the large intestine and causes attaching and effacing lesions (AE). Most of the genes involved in the formation of AE lesions are encoded within a chromosomal pathogenicity island termed the Locus of Enterocyte Effacement (LEE). The LysR-like transcriptional factor QseA regulates the LEE by binding directly to the regulatory region of ler. Here, we performed transcriptome analyses comparing WT EHEC and the isogenic qseA mutant in order to elucidate the extent of QseA’s role in gene regulation in EHEC. The following results compare genes that were up-regulated and down-regulated ! 2-fold in the qseA mutant strain compared to the WT strain. At mid-exponential growth, 222 genes were up-regulated and 1874 were downregulated. At late-exponential growth, a total of 55 genes were up-regulated and 605 genes were down-regulated. During mid-exponential growth, QseA represses its own transcription, whereas during late-logarithmic growth, QseA activates expression of the LEE genes as well as non-LEE encoded effector proteins. During both growth phases, several genes carried in O-islands, were activated by QseA, whereas genes involved in cell metabolism were repressed. We also performed electrophoretic mobility shift assays, competition experiments, and DNAseI footprints, and the results suggested that QseA directly binds both the ler proximal and distal promoters, its own promoter, as well as promoters of genes encoded in EHEC-specific O-islands. Additionally, we mapped the transcriptional start site of qseA, leading to the identification of two promoter sequences. Taken together, these results indicate that QseA acts as a global regulator in EHEC, coordinating expression of virulence genes. Design of the study was to make the knockout of the qseA gene and compare the transcriptional response to that and the wild type.
Project description:Genes identified in ETEC E24377A after interaction with host cells (Caco-2) after time points of 30 min, 60 min and 120 min. Both the adherent and non-adherent cells were profiled as well as E24377a grown only in tissue culture media. Each time and sample has at least duplicate samples
Project description:We and others have previously shown that glomerular endothelial cells and podocytes express hypoxia-inducible transcription factors (HIFs). HIFs bind to hypoxia response elements in target genes, such as vascular endothelial growth factor, which is continually produced by podocytes throughout life. To further assess function of HIFs in podocyte biology, podocin-Cre mice were mated with floxed von Hippel-Lindau (VHL) mice to selectively delete VHL, a component of an E3 ligase complex responsible for degradation of HIFs in normoxia. We reasoned that cells lacking VHL would overexpress stable HIFs and upregulate hypoxia-responsive genes. Progeny from these crosses displayed two phenotypes, non-proteinuric with glomerular basement membrane (GBM) defects and proteinuric with GBM defects and end-stage renal failure at ~6 months of age. Gene changes associated with the mild, non-proteinuric phenotype were studied using isolated glomeruli from wildtype and Pod-Cre fVHL mice. At 4 weeks of age, urine was collected and urinary albumin was quantified by Albuwell elisa from Pod-Cre fVHL litters. At 6 weeks of age, glomeruli from 3 wildtype littermate controls and 3 non-proteinuric Pod-Cre fVHL mice were collected using the magnetic bead method. RNA was extracted and hybridized to Affymetrix microarrays.