Project description:Purpose of study was to investigate whole genome expression changes of a strain with deletion of the two-component system TctD-TctE and determine genes dysregulate relative to the parental wildtype to gain insight into possible regulatory targets of TctD-TctE. TctD-TctE is a two-component system in Pseudomonas aeruginosa that responds to and regulates uptake of tricarboxylic acids such as citric acid. It accomplishes this through derepression of the porin encoding the gene opdH, thereby regulating influx of citrate metabolites from the surrounding environment. Deletion of the tctED operon (ΔtctED) resulted in a reduced growth phenotype when citric acid is present in media. In the ΔtctED strain the presence of citric acid was found to have an inhibitory effect on growth. When the alternative carbon source arginine was present, wildtype levels of growth could not be restored. Static cultures of ΔtctED had low cell density in the presence of citric acid but maintained the same levels of biofilm formation compared to conditions when no citric acid was present. This suggests a dysregulation of biofilm formation in the presence of citric acid. In the ΔtctED strain there was also greater accumulation of tobramycin within the biofilm compared to the PA14 wildtype strain. Additionally, analysis of whole-genome expression found that multiple metabolic genes were dysregulated in ΔtctED. Here it is concluded that TctD-TctE is involved in biofilm tolerance to tobramycin in the presence of citrate metabolites.
Project description:Infection of Pseudomonas donghuensis HYS strain and its fur deletion mutant in Caenorhabditis elegans was conducted to assess changes in the expression profile of Caenorhabditis elegans, and potential core virulence factors were identified by measuring the gene expression levels of the HYS colonizing the nematode's intestine. Preliminary studies indicate that P. donghuensis HYS exhibits significant toxicity towards Caenorhabditis elegans, yet the underlying mechanisms of this pronounced toxicity remain unclear. Previous work identified several virulence factors contributing to the toxicity of HYS through detection and functional validation; however, the molecular mechanisms responsible for its strong toxicity have not been elucidated. Therefore, we aim to analyze the mechanisms underlying HYS's pronounced toxicity by examining the responses of infected Caenorhabditis elegans. The Ferric uptake regulator (Fur) is responsible for maintaining iron homeostasis in Gram-negative bacteria, and given that HYS possesses a greater iron uptake capacity than other common species in the same genus, such as Pseudomonas aeruginosa, we hypothesize that Fur may play a critical role in the strong toxicity exhibited by HYS. Consequently, we infected Caenorhabditis elegans with both HYS and its fur deletion mutant and analyzed the changes in the expression profile of Caenorhabditis elegans. We observed a significant reduction in toxicity following the deletion of fur, indicating that Fur regulates core virulence factors. To identify these core virulence factors, we conducted transcriptomic sequencing of the pathogenic bacteria under various conditions and performed a screening for virulence factors.
Project description:To determine what genes play a role in virulence of global regulator cbrA. RNA was isolated from 3 biological repeats of PA14 cbrA deletion mutant and 3 biological repeats of Pseudomonas P14 parent strain grown on BM2 swarming plates. RNA was isolated from the edge of the dendritic clonies of PA14 and the entire colony on the non-swarming cbrA mutant.
Project description:Analysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in minimal medium with glucose as carbon source (M9G). SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF in Pseudomonas aeruginosa. Deletion of the ECF sigma factor sigX gene provide insights into the SigX role in several virulence and biofilm- related phenotypes in Pseudomonas aeruginosa. To better understand the cellular function of SigX, a deletion mutant of the sigX gene (PAOSX) was generated and its expression profile was compared with parental strain Pseudomonas aeruginosa H103. To this end, H103 and a sigX mutant were cultured in M9G, in which their growth are similar. Three independant biological replicate were taken for the RNA extraction and hybridization on affymetrix array in the middle of the exponential growth phase.
Project description:Analysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in minimal medium with glucose as carbon source (M9G). SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF in Pseudomonas aeruginosa. Deletion of the ECF sigma factor sigX gene provide insights into the SigX role in several virulence and biofilm- related phenotypes in Pseudomonas aeruginosa.
Project description:The bacterial type III secretion system (TTSS) is dedicated to directly effect host cell pathways by pathogens. In order to identify the key host responses of the various parts of the TTSS, we utilized expression profiling of a lung pneumocytes cell line, A549, exposed to various isogenic mutant strains of Pseudomonas aeruginosa PAK. We have devised a novel filtering method to isolate the key responses to the effector proteins as well as the translocation machinery. Individually, the effector proteins elicited host responses that were consistent with the known function of each, many of which either regulated, or are regulated by the cell cycle. However, our analysis has shown that the effector proteins elicit a distinct host expression pattern when present in combination, suggesting that these proteins function in synergy. Furthermore, the host transcriptional response to the translocation complex involved genes that are involved in remodeling of the plasma membrane, suggesting that the host cell is able to sense the protrusion of the TTSS machinery. This study shows that the individual components of the TTSS define an integrated system and that a systems biology approach is required to fully understand the complex interplay between pathogen and host. Keywords = Pseudomonas aeruginosa Keywords = A549 human lung carcinoma cell line Keywords = cystic fibrosis Keywords: repeat sample
Project description:Pseudomonas aeruginosa is a gram negative pathogen that infects acute wounds such as third degree skin injury and chronic wounds such as diabetic ulcers. Within infection sites, this pathogen exists in specific structures termed as biofilms. Biofilms contribute to enhanced resistance of microorganisms to the host defense and antibiotic treatments. Flagella, pilli and chaperon usher pathway (cup) fimbriae provide initial attachment to host tissue during biofilm development. pvcA-D operon codes for proteins which synthesize a secondary metabolite called paerucumarin. Paerucumarin is an isonitrile functionalized cumarin which is not extensively analyzed. We recently showed that paerucumarin enhances the expression of cup genes and biofilm development. We hypothesize that besides the cup genes pvcA-D operon regulates other P. aeruginosa genes. To test this hypothesis, we compared the transcriptome of P. aeruginosa strain MPAO1 with its pvcA mutant (MPAO1/pvcA). In comparison with MPAO1, 53 genes were differentially expressed in pvcA which included 19 up-regulated and 34 down-regulated genes. Functional characterization of differentially expressed genes indicated that 20 of these genes have been reported as iron regulated genes. Real time PCR confirmed these results and indicated that the expression of pvcAD operon is iron independent. However traditional chrome azurol S (CAS) iron binding assay showed that paerucumarin binds iron either within supernatants of MPAO1 or in solution. In addition, exogenously added paerucumarin enhances the expression of iron repressed genes pvdS and pvdA. Similarly, the level of pvdS gene expression in MPAO1?pvcA was significantly reduced as compared to MPAO1. Further analysis confirmed that paerucumarin binds iron in MPAO1 but does not deliver it inside the cell. The growth of a PAO1 double mutant strain (?pvdD?pchA), defective in iron scavenging systems, grown in iron deficient medium was restricted. However, the growth was restricted even further upon addition of exogenous paerucumarin to bacterial cultures. These results suggest that paerucumarin chelates iron but does not function as an iron scavenging system.
Project description:The goals of this study are to compare Next-Generation-Sequecing (NGS)-derived genome-wide occupancy of H3 and H4 acetylation in S. cerevisiae wildtype and gds1 deletion strain.
Project description:Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for high morbity in COPD, burns , implanted medical devices and cystic fibrosis. Pseudomonas aeruginosa is a problematic colonizer of the human lung. P. aeruginosa produces a phospholipase C (PlcH) that degrades choline-containing lipids such as phosphatidylcholine and sphingomylein that are found in lung surfactant and in host membranes. In this study, we analyzed gene expression in mutants defective in PlcH production (delta-plcH and delta-gbdR) and the wild type when growing in medium with lung surfactant. Pseudomonas aeruginosa was cultured in liquid cultures with aeration in a defined medium with Survanta, a lung surfactant replacement. Cultures were harvested during mid-exponential phase, and RNA was isolated for microarray analysis. The P. aeruginosa strain PAO1 wild type gene expression was compared to expression profiles from delta-gbdR and delta-plcHR deletion mutants, two mutants defective in PlcH production.