Andreas Dötsch

Publications (16) View all

• Article: Quantitative contribution of target alteration and decreased drug accumulation to Pseudomonas aeruginosa fluoroquinolone resistance.

  Sebastian Bruchmann, Andreas Dötsch, Bianka Nouri, Iris F Chaberny, Susanne Häussler
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  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 contribution of quinolone target alteration as well as the expression of efflux pumps to fluoroquinolone resistance in Pseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance determining regions (QRDR) of gyrA, gyrB and parC, and inactivated the efflux regulator-genes to overexpress the corresponding MDR efflux pump. We then introduced the respective mutations in the PA14 reference strain singularly and in various combinations. Whereas the combined inactivation of two efflux-regulator-encoding genes did not lead to higher resistance levels as compared to the inactivation of only one efflux-regulator-encoding gene; the combination of mutations leading to an 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 implicate that there are additional mechanisms to increase ciprofloxacin resistance in isolates with mutations in the QRDRs, which are independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN and/or MexXY-OprM efflux pumps.
  Antimicrobial Agents and Chemotherapy 12/2012; · 4.84 Impact Factor
• Article: The peptide chain release factor methyltransferase PrmC is essential for pathogenicity and environmental adaptation of Pseudomonas aeruginosa PA14.

  Christian Pustelny, Stephan Brouwer, Mathias Müsken, Agata Bielecka, Andreas Dötsch, Manfred Nimtz, Susanne Häussler
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  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.
  Environmental Microbiology 11/2012; · 5.84 Impact Factor
• Article: Mutation in Elongation Factor G Confers Resistance to the Antibiotic Argyrin in the Opportunistic Pathogen Pseudomonas aeruginosa.

  Piotr Bielecki, Peer Lukat, Kristina Hüsecken, Andreas Dötsch, Heinrich Steinmetz, Rolf W Hartmann, Rolf Müller, Susanne Häussler
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  ABSTRACT: The natural myxobacterial product argyrin is a cyclic peptide exhibiting immunosuppressive activity as well as antibacterial activity directed against the highly intrinsically resistant opportunistic pathogen Pseudomonas aeruginosa. In this study, we used whole-genome sequencing technology as a powerful tool to determine the mode of action of argyrin. Sequencing of argyrin-resistant P. aeruginosa isolates selected in vitro uncovered six point mutations that distinguished the resistant mutants from their susceptible parental strain. All six mutations were localized within one gene: fusA1, which encodes for the elongation factor EF-G. After the reintroduction of selected mutations into the susceptible wild type, the strain became resistant to argyrin. Surface plasmon resonance experiments confirmed the interaction of argyrin A with FusA1. Interestingly, EF-G has been previously shown to be the target of the anti-Staphylococcus antibiotic fusidic acid. Mapping of the mutations onto a structural model of EF-G revealed that the mutations conveying resistance against argyrin were clustered within domain III on the side opposite to that involved in fusidic acid binding, thus indicating that argyrin exhibits a new mode of protein synthesis inhibition. Although no mutations causing argyrin resistance have been found in other genes of P. aeruginosa, analysis of the sequence identity in EF-G and its correlation with argyrin resistance in different bacteria imply that additional factors such as uptake of argyrin play a role in the argyrin resistance of other organisms.
  ChemBioChem 09/2012; · 3.94 Impact Factor
• Source

  Available from: Stephane Plaisance

  Article: Global regulation of gene expression by OxyR in an important human opportunistic pathogen.

  Qing Wei, Phu Nguyen Le Minh, Andreas Dötsch, Falk Hildebrand, Warunya Panmanee, Ameer Elfarash, Sebastian Schulz, Stéphane Plaisance, Daniel Charlier, Daniel Hassett, Susanne Häussler, Pierre Cornelis
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  ABSTRACT: Most bacteria control oxidative stress through the H(2)O(2)-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ∼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.
  Nucleic Acids Research 01/2012; 40(10):4320-33. · 8.03 Impact Factor
• Source

  Available from: Andreas Dötsch

  Article: The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing.

  Andreas Dötsch, Denitsa Eckweiler, Monika Schniederjans, Ariane Zimmermann, Vanessa Jensen, Maren Scharfe, Robert Geffers, Susanne Häussler
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  ABSTRACT: In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5'-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.
  PLoS ONE 01/2012; 7(2):e31092. · 4.09 Impact Factor