ABSTRACT: Genome-wide transcriptional analysis of flagellin induced reprogramming in mouse corneal epithelial cells in response to Pseudomonas aeruginosa
Project description:We previously showed that pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against pathogenic microbes by reprogramming gene expression. To date, there was no genome-wide cDNA array to detect full scale of flagellin mediated reprogramming of gene expression in mucosal surface epithelial cells. Taking advantage of readily accessible, easily procurable epithelial cell population, this study is the first report to use genome-wide cDNA microarray approach to document genes associated with flagellin-induced protection against Pseudomonas aeruginosa infection in corneal epithelial cells (CECs). Total RNA obtained from isolated mouse corneal epithelial cells of the control (cells scrapped off from the corneas without infection), Pseudomonas aeruginosa infected (6 h post infection) and flagellin pretreated (24 h), followed by Pseudomonas aeruginosa infection (6 h).
Project description:We previously showed that pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against pathogenic microbes by reprogramming gene expression. To date, there was no genome-wide cDNA array to detect full scale of flagellin mediated reprogramming of gene expression in mucosal surface epithelial cells. Taking advantage of readily accessible, easily procurable epithelial cell population, this study is the first report to use genome-wide cDNA microarray approach to document genes associated with flagellin-induced protection against Pseudomonas aeruginosa infection in corneal epithelial cells (CECs).
Project description:Recombinant protein of Pseudomonas aeruginosa hook protein FlgE was added to cultured human corneal epithelial cell line for 4 hours and the mRNA expression profiling was performed using Agilent 8*60K array and dual labeling.
Project description:Recombinant protein of Pseudomonas aeruginosa hook protein FlgE was added to cultured human corneal epithelial cell line for 4 hours and the mRNA expression profiling was performed using Agilent 8*60K array and dual labeling. Three triplicates were included for FlgE treatment and PBS control respectively, thus producing three pairs of samples for array, namely E1PBS1, E2PBS2, E3PBS3.
Project description:Purpose: To determine effects of arsenic on gene expression in polarized primary human bronchial epithelial (HBE) cells and impact on transcriptional response to Pseudomonas aeruginosa infection Methods: mRNA profiles of HBE cells from 6 donors exposed to 0, 5, 10 or 50 ug/L total arsenic +/- Pseudomonas aeruginosa (48 samples) were generated using Illumina sequencing, aligned in CLC Genomics workbench and analyzed for DE in EdgeR Findings: 20-30 million reads were mapped per sample and transcripts were identifed that were significantly differentially expressed in response to arsenic and Pseudomonas aeruginosa
Project description:The purpose of the study is to compare the transcriptomic profile of the airway epithelial cell line Calu-3 in reponse to Pseudomonas aeruginosa virulence factor flagellin. CF is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR gene in Calu-3 cells was knockdown by CRISPR-Cas9 (TS) and a control cell line (CTL) was aslo generated using a non-targeting guide RNA. CTL and CFTR KD Calu3-cells were grown on Transwell filters, polarized at the air-liquid interface and exposed to flagellin for 6 hours. We considered 2 non-stimulated (ND) conditions: CTL-NS and TS-NS, and 2 conditions stimulated with flagellin (Flag): CTL-Flag and TS-Flag.
Project description:Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of Pseudomonas aeruginosa PAO1. The absence of phosphorylation in the conserved T27 and S28 residues of flagellin FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. Flagellin phosphomutants had increased levels and activities of type 2 secretome proteins. This occurs by a possible mechanism affecting the secretion efficiency of T2SS machinery. Flagellin phosphomutants also formed reduced biofilm at 24h and had delayed biofilm dispersal under static and dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in the levels of type 2 secretome was accompanied by reduced biofilm formation in the flagellin phosphomutants. Altogether, we found a system of phosphorylation that co-ordinately regulates surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.
Project description:Immune responses in plants are triggered in part by conserved microbe-associated molecular pattern (MAMP) molecules such as bacterial flagellin. Upon MAMP perception, plants rapidly turn on the induction of numerous defense-related genes. We have identified a novel type of plant innate immunity elicitor, protease IV from Pseudomonas aeruginosa. Genome-wide transcriptomic profiles obtained with Affymetrix Arabidopsis ATH1 GeneChips® of 10-day old Arabidopsis seedlings treated with 20 nM purified protease IV for 1 hour were compared to published bacterial flagellin- and oligogalacturonide-triggered responses. We used microarrays to characterize the global transcriptomic changes in Arabidopsis seedlings upon protease IV treatment.
Project description:In our study, we used a mouse model infected with Pseudomonas aeruginosa (PA) to provoke a painful, sight-threatening corneal infection. FACS sorted WT and Gal-8-/- neutrophils of PA infected corneas were processed for next-generation transcriptomic RNA sequencing (RNA-seq).
Project description:Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (MMP-7) and stromelysin-2 (MMP-10), two matrix metalloproteinases induced by acute P. aeruginosa pulmonary infection. Extraction of Differential Gene Expression (EDGE) analysis of gene expression changes in P. aeruginosa infected organotypic tracheal epithelial cell cultures from wildtype, Mmp7-/-, and Mmp10-/- mice identified 2,089 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to pseudomonas infection and show that a global genomics strategy can be used to assess MMP function. Experiment Overall Design: C57Bl6, Mmp7-/- and Mmp10-/- mouse epithelium at an organotypic air liquid interface culture was exposed to Pseudomonas aeruginosa for 1 and 24 h. RNA was collected from these samples as well as uninfected 0 h and assessed for expression changes using Affymetrix Mouse 430 2.0 Arrays. Triplicate samples were processed for each genotype at each time point.
