Project description:Pseudomonas aeruginosa is a predominant pathogen in chronic lung infections in individuals with cystic fibrosis (CF). Epidemic strains of P. aeruginosa, such as the Liverpool Epidemic Strain (LES), are capable of transferring between CF patients and have been associated with increased hospital visits and antibiotic treatments. We used label-free quantitative proteomics to compare the laboratory strain PAO1, beta-lactam resistant isolate LESB58, and beta-lactam susceptible isolate LESlike1 and their responses to three beta-lactams (aztreonam, carbenicillin, piperacillin), the aminoglycoside tobramycin, and hydrogen peroxide. Across all samples, we identified 3019 proteins with a minimum of two peptides. We found that LESB58 showed a large response to treatment with the beta-lactam carbenicillin, with 644 proteins significantly increased in abundance and 590 proteins significantly decreased in abundance (Students t-test, p≤0.05, FDR=0.05, S0=1). Proteomic characterization of an additional beta-lactam resistant isolate, LES431, exposed to carbenicillin showed that this response was shared by both isolates. Part of the response to carbenicillin in LESB58 included an increase in abundance in proteins involved in cell wall synthesis and division.
Project description:Surfing motility is a complex adaptation that is different from swarming motility and requires the stringent stress response in Pseudomonas aeruginosa LESB58 Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick and sticky mucus that can lead to progressive airway obstruction. The glycoprotein mucin is the major macromolecular component of mucus. Recently, we identified that the presence of mucin induced a rapid surface adaptation termed surfing motility in motile bacteria. P. aeruginosa, the main colonizing pathogen in CF employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. Here, RNA-Seq was used to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain) via transcriptional profiling. As the stringent response is regulated by relA and spoT, we created a double knockout of these genes in LESB58 to study the impact of these stress regulators on surfing motility using RNA-Seq.
Project description:The ParS/ParR two component regulatory system plays important roles for multidrug resistance in Pseudomonas aeruginosa. In this study we report RNA-seq analyses of the transcriptomes of P. aeruginosa PAO1 wild type and par mutants growing in a minimal medium containing 2% casamino acids. This has allowed the quantification of PAO1 transcriptome, and further defines the regulon that is dependent on the ParS/ParR system for expression. Our RNA-seq analysis produced the first estimates of absolute transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished the expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to affecting drug resistance genes, transcripts of quorum sensing genes (rhlIR and pqsABCDE-phnAB), were significantly up-regulated in both parS and parR mutants. Consistent with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of par genes also lead to overproduction of phenazines and increased swarming motility, consistent with the up-regulation of quorum sensing genes. Our results established a link among ParS/ParR, MexEF-OprN and quorum sensing in Pseudomonas aeruginosa. Based on these results, we propose a model to illustrate the relationship among these regulatory systems in P. aeruginosa. A total of 9 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas aeruginosa PAO1 wild type strain (3 replicates); Pseudomonas aeruginosa parS mutant (3 replicates); Pseudomonas aeruginosa parR mutant (3 replicates).
Project description:Pseudomonas aeruginosa (Pa) is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. The outer membrane composition of Pa restricts antibiotic entry and determines virulence. For efficient outer membrane protein biogenesis, the BAM complex and chaperones like Skp and SurA are crucial. Deletion mutants of bamB, bamC and the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects were associated with a lack of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the conditional deletion of surA in a multidrug-resistant bloodstream isolate re-sensitized the strain to antibiotic treatment. Mass spectrometry revealed striking alterations in the outer membrane composition. Thus, SurA of Pa is important for the insertion of many porins, type V secretion systems, TonB-dependent receptors, proteins involved in LPS transport and BAM complex components. Therefore, SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and sensitizes multidrug-resistant strains to antibiotics.
Project description:Methylrhodomelol (1) is a bromophenol from the red alga Vertebrata lanosa (L.) T.A.Christensen that has been associated with antimicrobial properties. Aim of the current study was therefore, to assess the antimicrobial potential of this compound in more detail against the gram-negative pathogen Pseudomonas aeruginosa. 1 exerted weak bacteriostatic activity against different strains when grown in minimal medium, whereas other phenolics were inactive. In addition, 1 (35 and 10 µg/mL) markedly enhanced the susceptibility of multidrug resistant P. aeruginosa towards the aminoglycoside gentamicin, while it did not affect the viability of Vero kidney cells up to 100 µM. Finally, pyoverdine release was reduced in bacteria treated at sub-inhibitory concentration, but no effect on other virulence factors was observed. Transcriptome analysis of treated versus untreated P. aeruginosa indicated an interference of 1 with bacterial carbon and energy metabolism, which was corroborated by RT-qPCR and decreased ATP-levels in treated bacteria.
Project description:Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen, with chronic infections often associated with biofilms that enhance antibiotic resistance. This study investigates the uncharacterized gene PA3049, which is upregulated under biofilm conditions, to determine its role in infection, biofilm formation, and antimicrobial tolerance. o Using bioinformatics, infection models, and molecular microbiology, we determined that PA3049 contributed to biofilm establishment both in vitro and in high-validity infection models. We also identified its role in bacterial survival under sub-inhibitory concentrations of antibiotics and its impact on pyocyanin production. Proteomic analysis revealed that PA3049 upregulates the R2-type pyocin cluster, which drives explosive cell lysis and extracellular DNA (eDNA) release during early stages of P. aeruginosa biofilm development. Additionally, PA3049 interacts with PA0486, an uncharacterized Ser/Thr protein kinase implicated in pyocyanin production and bacterial killing, suggesting a putative in vivo mechanism of action. o Renamed as the Biofilm antibiotic tolerance Regulator (BatR), PA3049 emerges as a key player in P. aeruginosa biofilm maintenance and resistance. These findings provide new insights into bacterial biofilm dynamics and highlight two previously uncharacterized genes with potential implications for combating multidrug-resistant infections