http://hdl.handle.net/10033/223075 Wed, 19 Jun 2013 16:53:25 GMT 2013-06-19T16:53:25Z http://hdl.handle.net/10033/293149 Title: The peptide chain release factor methyltransferase PrmC is essential for pathogenicity and environmental adaptation of Pseudomonas aeruginosa PA14. Authors: Pustelny, Christian; Brouwer, Stephan; Müsken, Mathias; Bielecka, Agata; Dötsch, Andreas; Nimtz, Manfred; Häussler, Susanne Abstract: Pseudomonas aeruginosa pathogenicity and its capability to adapt to multiple environments are dependent on the production of diverse virulence factors, controlled by the sophisticated quorum sensing (QS) network of P. aeruginosa. To better understand the molecular mechanisms that underlie this adaptation we searched for novel key regulators of virulence factor production by screening a PA14 transposon mutant library for potential candidates acting downstream of the unique 2-alkyl-4-quinolone (AQ) QS system of P. aeruginosa. We focused the work on a protein named HemK with high homology to PrmC of Escherichia coli displaying a similar enzymatic activity (therefore also referred to as PrmC). In this study, we demonstrate that PrmC is an S-adenosyl-l-methionine (AdoMet)-dependent methyltransferase of peptide chain release factors (RFs) essential for the expression of several virulence factors, such as pyocyanin, rhamnolipids and the type III-secreted toxin ExoT. Furthermore, the PA14_prmC mutant strain is unable to grow under anoxic conditions and has a significantly reduced pathogenicity in the infection model Galleria mellonella. Along with transcriptomic and proteomic analyses, the presented data indicate that the methylation of RFs in P. aeruginosa seems to have a global effect on cellular processes related to the virulence of this nosocomial pathogen. Fri, 01 Feb 2013 00:00:00 GMT http://hdl.handle.net/10033/293149 2013-02-01T00:00:00Z http://hdl.handle.net/10033/279871 Title: Recycling of Peptidyl-tRNAs by Peptidyl-tRNA Hydrolase Counteracts Azithromycin-Mediated Effects on Pseudomonas aeruginosa. Authors: Gödeke, Julia; Pustelny, Christian; Häussler, Susanne Abstract: Acute and chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa pose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronic P. aeruginosa infections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility in P. aeruginosa seem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of the P. aeruginosa peptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon in rhlR also explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on the P. aeruginosa proteome and possibly also on the protein expression profiles of other bacterial pathogens. Mon, 01 Apr 2013 00:00:00 GMT http://hdl.handle.net/10033/279871 2013-04-01T00:00:00Z http://hdl.handle.net/10033/279512 Title: Ex vivo transcriptional profiling reveals a common set of genes important for the adaptation of Pseudomonas aeruginosa to chronically infected host sites. Authors: Bielecki, Piotr; Komor, Uliana; Bielecka, Agata; Müsken, Mathias; Puchałka, Jacek; Pletz, Mathias W; Ballmann, Manfred; Martins dos Santos, Vítor A P; Weiss, Siegfried; Häussler, Susanne Abstract: The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P.  aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm-associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases. Fri, 01 Feb 2013 00:00:00 GMT http://hdl.handle.net/10033/279512 2013-02-01T00:00:00Z http://hdl.handle.net/10033/275612 Title: Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance. Authors: Bruchmann, Sebastian; Dötsch, Andreas; Nouri, Bianka; Chaberny, Iris F; Häussler, Susanne Abstract: Quinolone antibiotics constitute a clinically successful and widely used class of broad-spectrum antibiotics; however, the emergence and spread of resistance increasingly limits the use of fluoroquinolones in the treatment and management of microbial disease. In this study, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, and parC and inactivated the efflux regulator genes so as to overexpress the corresponding multidrug resistance (MDR) efflux pumps. We then introduced the respective mutations into the reference strain PA14 singly and in various combinations. Whereas the combined inactivation of two efflux regulator-encoding genes did not lead to resistance levels higher than those obtained by inactivation of only one efflux regulator-encoding gene, the combination of mutations leading to increased efflux and target alteration clearly exhibited an additive effect. This combination of target alteration and overexpression of efflux pumps was commonly observed in clinical P. aeruginosa isolates; however, these two mechanisms were frequently found not to be sufficient to explain the level of fluoroquinolone resistance. Our results suggest that there are additional mechanisms, independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and/or MexXY-OprM efflux pump, that increase ciprofloxacin resistance in isolates with mutations in the QRDRs. Fri, 01 Mar 2013 00:00:00 GMT http://hdl.handle.net/10033/275612 2013-03-01T00:00:00Z