Project description:Using an integrated model system for reproducible growth of biofilms, a JPIAMR-funded consortium of researchers* studied the expressed proteome of P. aeruginosa strain MPAO1 under i) planktonic growth, and ii) biofilm formation conditions. The model system included, as a first step, the sequencing and de novo assembly of the complete genome of this opportunistic human pathogen that belongs to the notorious group of Gram-negative ESKAPE pathogens. MPAO1 is also the parental strain for the widely used transposon (Tn) mutant library from the University of Washington. The complete MPAO1 genome sequence turned out to harbor several deletions and insertions compared to the PAO1-UW reference genome including numerous MPAO1-unique genes. As a second step in the model system, a biofilm flow cell based on poly (dimethylsiloxane) (PDMS) was designed to reproducibly study and identify known and novel genes related to biofilm growth and antibiotic resistance (ABR) from the Tn mutant collection. With the complete genome as optimal basis, publicly available TnSeq data were reanalyzed to identify known and novel essential genes. Furthermore, shotgun proteomics data was generated uncovering 1530 (planktonic) and 1728 (biofilm) expressed proteins, respectively, resulting in the identification of 1922 (33.1%) of the 5799 annotated P. aeruginosa MPAO1 proteins. They included proteins known to be differentially expressed during biofilm formation, and proteogenomic evidence for proteins uniquely encoded by MPAO1 as well as novel proteins.
Project description:Transcriptional profiling in Pseudomonas aeruginosa MPAO1 and its glyoxylate shunt gens transposon insertion mutants (aceA and glcB) was perfomed using microarray analysis with or without 1mM PQ treatment. Based on the gene expression, we investigated role of glyoxylate shunt in P. aeruginosa and found out pathway related with glyoxylate shunt under oxidative stress.
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: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
Project description:Wound infections are traditionally thought to occur when microbial burden exceeds the innate clearance capacity of host immune system. Here we introduce the idea that the wound environment itself plays a significant contributory role to wound infection. We developed a clinically relevant murine model of soft tissue infection to explore the role of activation of microbial virulence in response to tissue factors as a mechanism by which pathogenic bacteria cause wound infections. Mice underwent abdominal skin incision and light muscle injury with a crushing forceps versus skin incision alone followed by topical inoculation of Pseudomonas aeruginosa. Pseudomonas aeruginosa whole genome transcriptional profiling demonstrated that fascia induced the activation of multiple genes responsible for the synthesis of the iron scavenging protein pyochelin. Ex-vivo murine fascia homogenates were prepared and Pseudomonas aeruginosa MPAO1 was incubated with an inoculum of the fascia homogenate solution. Pseudomonas aeruginosa MPAO1 incubated under the same condtions without the homogenate was used as the control group. Three biological replicates in each group was used.