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Pohl S.,Helmholtz Center for Infection Research | Pohl S.,TWINCORE GmbH | Klockgether J.,Hannover Medical School | Eckweiler D.,Helmholtz Center for Infection Research | And 9 more authors.
FEMS Microbiology Letters | Year: 2014

Up to 20% of the chromosomal Pseudomonas aeruginosa DNA belong to the so-called accessory genome. Its elements are specific for subgroups or even single strains and are likely acquired by horizontal gene transfer (HGT). Similarities of the accessory genomic elements to DNA from other bacterial species, mainly the DNA of γ- and β-proteobacteria, indicate a role of interspecies HGT. In this study, we analysed the expression of the accessory genome in 150 clinical P. aeruginosa isolates as uncovered by transcriptome sequencing and the presence of accessory genes in eleven additional isolates. Remarkably, despite the large number of P. aeruginosa strains that have been sequenced to date, we found new strain-specific compositions of accessory genomic elements and a high portion (10-20%) of genes without P. aeruginosa homologues. Although some genes were detected to be expressed/present in several isolates, individual patterns regarding the genes, their functions and the possible origin of the DNA were widespread among the tested strains. Our results demonstrate the unaltered potential to discover new traits within the P. aeruginosa population and underline that the P. aeruginosa pangenome is likely to increase with increasing sequence information. © 2014 Federation of European Microbiological Societies.


Blanka A.,Twincore GmbH | Schulz S.,Twincore GmbH | Schulz S.,Helmholtz Center for Infection Research | Eckweiler D.,Twincore GmbH | And 12 more authors.
Journal of Bacteriology | Year: 2014

Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative σ factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa. © 2014, American Society for Microbiology.


Ahmad I.,Karolinska Institutet | Wigren E.,Karolinska Institutet | Le Guyon S.,Karolinska Institutet | Vekkeli S.,Karolinska Institutet | And 11 more authors.
Molecular Microbiology | Year: 2013

Summary: The ubiquitous second messenger c-di-GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c-di-GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL-like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT-29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL-like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c-di-GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S.typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function. © 2013 John Wiley & Sons Ltd.


Bielecki P.,Yale University | Bielecki P.,TWINCORE GmbH | Jensen V.,TWINCORE GmbH | Schulze W.,TWINCORE GmbH | And 11 more authors.
Nucleic Acids Research | Year: 2015

Two-component systems (TCS) serve as stimulus-response coupling mechanisms to allow organisms to adapt to a variety of environmental conditions. The opportunistic pathogen Pseudomonas aeruginosa encodes for more than 100 TCS components. To avoid unwanted cross-talk, signaling cascades are very specific, with one sensor talking to its cognate response regulator (RR). However, cross-regulation may provide means to integrate different environmental stimuli into a harmonized output response. By applying a split luciferase complementation assay, we identified a functional interaction of two RRs of the OmpR/PhoB subfamily, namely PhoB and TctD in P.aeruginosa. Transcriptional profiling, ChIP-seq analysis and a global motif scan uncovered the regulons of the two RRs as well as a quadripartite binding motif in six promoter regions. Phosphate limitation resulted in PhoB-dependent expression of the downstream genes, whereas the presence of TctD counteracted this activation. Thus, the integration of two important environmental signals e.g. phosphate availability and the carbon source are achieved by a titration of the relative amounts of two phosphorylated RRs that inversely regulate a common subset of genes. In conclusion, our results on the PhoB and TctD mediated two-component signal transduction pathways exemplify how P.aeruginosa may exploit cross-regulation to adapt bacterial behavior to complex environments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


Blanka A.,TWINCORE GmbH | Duvel J.,TWINCORE GmbH | Duvel J.,Helmholtz Center for Infection Research | Dotsch A.,TWINCORE GmbH | And 9 more authors.
Science Signaling | Year: 2015

Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3',5'-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5' untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses.


Pustelny C.,Helmholtz Center for Infection Research | Pustelny C.,TWINCORE GmbH | Komor U.,Helmholtz Center for Infection Research | Pawar V.,Helmholtz Center for Infection Research | And 15 more authors.
Infection and Immunity | Year: 2015

The recent finding that high numbers of strict anaerobes are present in the respiratory tract of cystic fibrosis (CF) patients has drawn attention to the pathogenic contribution of the CF microbiome to airway disease. In this study, we investigated the specific interactions of the most dominant bacterial CF pathogen, Pseudomonas aeruginosa, with the anaerobic bacterium Veillonella parvula, which has been recovered at comparable cell numbers from the respiratory tract of CF patients. In addition to growth competition experiments, transcriptional profiling, and analyses of biofilm formation by in vitro studies, we used our recently established in vivo murine tumor model to investigate mutual influences of the two pathogens during a biofilm-associated infection process. We found that P. aeruginosa and V. parvula colonized distinct niches within the tumor. Interestingly, significantly higher cell numbers of P. aeruginosa could be recovered from the tumor tissue when mice were coinfected with both bacterial species than when mice were monoinfected with P. aeruginosa. Concordantly, the results of in vivo transcriptional profiling implied that the presence of V. parvula supports P. aeruginosa growth at the site of infection in the host, and the higher P. aeruginosa load correlated with clinical deterioration of the host. Although many challenges must be overcome to dissect the specific interactions of coinfecting bacteria during an infection process, our findings exemplarily demonstrate that the complex interrelations between coinfecting microorganisms and the immune responses determine clinical outcome to a much greater extent than previously anticipated. © 2015, American Society for Microbiology.


Khaledi A.,Helmholtz Center for Infection Research | Khaledi A.,TWINCORE GmbH | Schniederjans M.,Helmholtz Center for Infection Research | Schniederjans M.,TWINCORE GmbH | And 18 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2016

Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


Haussler S.,TWINCORE GmbH | Haussler S.,Helmholtz Center for Infection Research | Blanka A.,TWINCORE GmbH | VanHook A.M.,Science Signaling
Science Signaling | Year: 2015

This Podcast features an interview with Andrea Blanka and Susanne Häussler, authors of a Research Article that appears in the 14 April 2015 issue of Science Signaling, about a mutation that enhances the ability of a pathogenic strain of Pseudomonas aeruginosa to form biofilms. Biofilms are microbial communities that grow on biological and nonbiological surfaces. The microbes in biofilms are embedded in a complex matrix that protects them from antibiotics and from being destroyed by the immune system. P. aeruginosa is an opportunistic pathogen that is more difficult to treat when it forms a biofilm than when it exists as freeliving cells. Variants of P. aeruginosa that have an enhanced ability to form biofilms are frequently isolated from the chronically infected lungs of cystic fibrosis (CF) patients. Blanka et al. identified a mutation in a strain of CFassociated P. aeruginosa that affects lipid metabolism, leading to altered composition of the bacterial plasma membrane. This mutation caused the bacteria to constitutively produce the second messenger cdiGMP, which promotes biofilm formation.


Patent
Twincore GmbH and Cnrs | Date: 2013-03-22

The present invention relates to flavone derivatives and to compositions containing one or more of these flavone derivatives. The present invention further relates to flavone derivatives or compositions for use in the treatment and/or prevention of a viral infection, and to a method of preventing or treating these infections.


PubMed | Twincore GmbH
Type: Journal Article | Journal: Journal of bacteriology | Year: 2013

Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma () factors. The largest group of alternative factors is that of the extracytoplasmic function (ECF) factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa.

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