Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome of pvcA mutant as compared to parent strain of pseudomonas aeruginosa

ABSTRACT: 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. 4 biological replicates of each strain were randomly paired

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Uzma Qaisar

PROVIDER: E-GEOD-45973 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions. Transcriptional profiling of two independent biological replicates of Pseudomonas aeruginosa biofilms, as grown to 72 hours and used as inocula applied to the murine wounds, was performed. A principle components analysis (PCA) was used to provide an overview of the transcriptome data from the 28-day mouse wound scab, comparing the data to the biofilm inoculum, and to published reports of P. aeruginosa biofilm and planktonic samples. The analysis shows that the transcriptome of the mouse wound scab was distinct from the biofilm inoculum that was applied to the wound, demonstrating a shift in biofilm gene expression following 28 days of infection. We sought to characterize P. aeruginosa activity within biofilms in the mouse wound model by isolating and identifying mRNA from the biofilms used as inocula and from the wound scabs 28 days post infection.

Project description:P. aeruginosa is the leading cause of death in patients with cystic fibrosis patients and one of the most problematic bacterial pathogens responsible for hospital-acquired infections. This pathogen has a high capacity to form biofilms on inert and living surfaces. This lifestyle allows it to persist in various hospital niches or on medical device which become vectors of contamination. Chronic infections are extremely complicated to eradicate due to the remarkable antimicrobial resistance of biofilms leading to a persistence in the tissue and an immune system exhaustion. It is therefore becoming essential to understand the mechanisms of biofilm formation to find new therapeutic targets in order to develop effective antibiofilm strategies. We previously identified in P. aeruginosa PA01 biofilms an accumulation of a hypothetical protein named PA3731 and its deletion impacted the biofilm formation. Similarly, to PspA, a protein from the well-known Psp system of E. coli, PA3731 is a has a predicted structure mostly helical, a PspA/IM30 domain and was accumulated during an osmotic shock. In P. aeruginosa genome, PA3731 appears to form a cluster with 3 genes (PA3732 to PA3729) that we named BAC system for “Biofilm Associated Cluster”. Here we worked on the PA14 strain and focus our study on PA14_16140, the PA3732 homologue. Using a ∆16140 mutant and phenotypic approach, we confirmed the role of the BAC system in the virulence and biofilm formation. We added supplementary genes coding the BAC system and demonstrate that altogether they form an operonic structure regulates by RpoN. We get further insight the role PA14_16140 by proteomic quantitative approach revealing an accumulation of the BAC system proteins in ∆16140 biofilms suggesting its regulatory role of the bac operon. Moreover, we present here the first crystallographic structure of PA14_16140. To summarise, according to our studies, and although further analysis is still required, this newly discovered operon appears composed firstly of its regulator and then of a homologous PspA.

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Transcriptome of pvcA mutant as compared to parent strain of pseudomonas aeruginosa

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