Project description:The transcriptional regulator AmpR controls expression of the AmpC ß-lactamase in P. aeruginosa and other bacteria. Studies have demonstrated that in addition to regulating ampC expression, AmpR also regulates the expression of the sigma factor AlgT/U and the production of some quorum-sensing regulated virulence factors. In order to understand the ampR regulon, we compared the expression profiles of PAO1 and its isogenic ampR mutant, PAO∆ampR in the presence and absence of sub-MIC ß-lactam stress. The analysis demonstrates that the ampR regulon is much more extensive than previously thought, with the deletion of ampR affecting the expression of over 300 genes. Expression of an additional 207 genes are affected by AmpR when the cells are exposed to sub-MIC ß-lactam stress, indicating that the ampR regulon in P. aeruginosa is much more extensive than previously thought. An inframe deletion of ampR was generated in P. aeruginosa PAO1. The wild type and ampR mutant strains were grown to mid-log phase and subjected to sub-MIC ß-lactam exposure. Total RNA was isolated from 2-hour ß-lactam exposed and unexposed cells to monitor changes in gene expression arising due to loss of ampR in the presence and absence of ß-lactam exposure.
Project description:The transcriptional regulator AmpR controls expression of the AmpC ß-lactamase in P. aeruginosa and other bacteria. Studies have demonstrated that in addition to regulating ampC expression, AmpR also regulates the expression of the sigma factor AlgT/U and the production of some quorum-sensing regulated virulence factors. In order to understand the ampR regulon, we compared the expression profiles of PAO1 and its isogenic ampR mutant, PAO∆ampR in the presence and absence of sub-MIC ß-lactam stress. The analysis demonstrates that the ampR regulon is much more extensive than previously thought, with the deletion of ampR affecting the expression of over 300 genes. Expression of an additional 207 genes are affected by AmpR when the cells are exposed to sub-MIC ß-lactam stress, indicating that the ampR regulon in P. aeruginosa is much more extensive than previously thought.
Project description:Antimicrobial resistance (AMR) is a major challenge for global health. Multi-drug treatments can help to reduce AMR and be implemented through the sequential administration of antimicrobials (Roemhild and Schulenburg 2019). Such sequential treatments can achieve high efficacy through negative cellular hysteresis: the antibiotic-induced sensitization of bacterial cells towards a subsequently administered antibiotic (Roemhild et al. 2018). However, to date, the exact characteristics of antibiotic sensitization via negative hysteresis are unknown. Therefore, our study aimed at providing fundamental insights into the nature of hysteresis and its possible applicability to treatment, focusing on the high-risk human pathogen Pseudomonas aeruginosa. We found that negative hysteresis is robustly expressed across the species P. aeruginosa, particularly upon switches from β-lactam antibiotics to aminoglycosides, even if the β-lactam is administered at sub-MIC levels. We further characterized the molecular basis of hysteresis by a transcriptomic analysis of wildtype Pseudomonas PA-14 and a PA-14 CpxS T163P mutant that is described here. We demonstrate that the Cpx envelope stress response system is centrally involved in inducible cellular sensitization. Furthermore, negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions. Overall, our study reveals negative hysteresis to be associated with β-lactam-induced membrane stress, widely expressed across P. aeruginosa, thus identifying this phenomenon as a promising focus for effective antimicrobial therapy.
Project description:We report a next-generation sequencing of total RNA from Pseudomonas aeruginosa PAO1 grown in presence of rosmarinic acid (RA) 100mM. Data analysis in comparison with cells grown in absence of RA revealed that the plant compound RA induces a broad transcriptional response in this bacterium, quite similar to the quorum sensing response.
Project description:Gene expression of P. aerruginosa changes after short-term exposure to ciprofloxacin at sub-inhibitory concentrations but the effect of long-term exposure which select for the most fitted subpopulations is not known. We used microarrays to investigate the changes in gene expression of P.aeruginosa PAO1 and mutator (Î? mutS) after long-term evolution (94 daily passages) in LB in the presence and absence of ciprofloxacin Three different colonies from the ancestral populations of PAO1 and mutator (Î? mutS) as well as from the evolved populations (day 94) of each of the three lineages (A;B;C) in the presence or absence of ciprofloxacin at a concentration of 0.05 µg/ml were used for overnight cultures in LB and total RNA was extracted at OD600nm=1 and hybridized on P. aeruginosa Affymetrix chip.
Project description:The transcriptome of P. aeruginosa PAO1 in the presence of extracelluar 2-oxoglutarate at a concentration of 20 mM. We determined the transcriptional response of P. aeruignosa PAO1 to extracellular 2-oxoglutarate.
Project description:PAO1 was grown in sub-inhibitory ciprofloxacin (0.1x-, 0.3x-, 1x-MIC) until log phase. Microarrays were done on total RNA isolated from these cultures. Loop design and dye swapping were used.
Project description:Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, causing serious chronic infections. PA can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound PA isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and apply proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the PA genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.
Project description:The role of the intracellular signaling molecule diadenosine tetraphosphate (Ap4A) has not so far been investigated in Pseudomonas aeruginosa. To fill this gap, we performed RNA sequencing (RNA-seq) analysis to compare the transcriptome of the reference strain P. aeruginosa PAO1 and an isogenic deletion mutant (ΔapaH) which is deficient in the Ap4A hydrolysing enzyme ApaH and accumulates high intracellular levels of Ap4A.
