• Analysis of the organization and expression patterns of the convergent pseudomonas aeruginosa lasr/rsal gene pair uncovers mutual influence.

      Schinner, Silvia; Preusse, Matthias; Kesthely, Christopher; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-10-19)
      The two adjacent genes encoding the major Pseudomonas aeruginosa quorum sensing regulator, LasR, and its opponent, RsaL, overlap in their coding 3´ends and produce mRNA transcripts with long untranslated 3´ends that overlap with the sense transcripts of the gene on the opposing DNA strand. In this study, we evaluated whether the overlapping genes are involved in mutual regulatory events and studied interference by natural antisense transcripts. We introduced various gene expression constructs into a P. aeruginosa PA14 lasR/rsaL double deletion mutant, and found that although complementary RNA is produced, this does not interfere with the sense gene expression levels of lasR and rsaL and does not have functional consequences on down-stream gene regulation. Nevertheless, expression of lasR, but not of rsaL, was shown to be enhanced if transcription was terminated at the end of the respective gene so that no overlapping transcription was allowed. Our data indicate that the natural organization with a partial overlap at the 3´ends of the lasR/rsaL genes gives rise to a system of checks and balances to prevent dominant and unilateral control by LasR over the RsaL transcriptional regulator of opposing function.
    • A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa.

      Düvel, Juliane; Bertinetti, Daniela; Möller, Stefan; Schwede, Frank; Morr, Michael; Wissing, Josef; Radamm, Lena; Zimmermann, Bastian; Genieser, Hans-Gottfried; Jänsch, Lothar; et al. (2012-02)
      In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.
    • Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa.

      Bielecki, Piotr; Jensen, Vanessa; Schulze, Wiebke; Gödeke, Julia; Strehmel, Janine; Eckweiler, Denitsa; Nicolai, Tanja; Bielecka, Agata; Wille, Thorsten; Gerlach, Roman G; et al. (2015-07-27)
      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.
    • Detrimental Effect of Type I IFNs During Acute Lung Infection With Is Mediated Through the Stimulation of Neutrophil NETosis.

      Pylaeva, Ekaterina; Bordbari, Sharareh; Spyra, Ilona; Decker, Anna Sophie; Häussler, Susanne; Vybornov, Vadim; Lang, Stephan; Jablonska, Jadwiga; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Pseudomonas aeruginosa is an opportunistic multidrug-resistant pathogen, able to grow in biofilms. It causes life-threatening complications in diseases characterized by the up-regulation of type I interferon (IFN) signaling, such as cancer or viral infections. Since type I IFNs regulate multiple functions of neutrophils, which constitute the first line of anti-bacterial host defense, in this work we aimed to study how interferon-activated neutrophils influence the course of P. aeruginosa infection of the lung. In lungs of infected IFN-sufficient WT mice, significantly elevated bacteria load was observed, accompanied by the prominent lung tissue damage. At the same time IFN-deficient animals seem to be partly resistant to the infection. Lung neutrophils from such IFN-deficient animals release significantly lower amounts of neutrophil extracellular traps (NETs) and reactive oxygen species (ROS), as compared to WT neutrophils. Of note, such IFN-deficient neutrophils show significantly decreased capacity to stimulate biofilm formation by P. aeruginosa. Reduced biofilm production impairs in turn the survival of bacteria in a lung tissue. In line with that, treatment of neutrophils with recombinant IFN-β enhances their NETosis and stimulates biofilm formation by Pseudomonas after co-incubation with such neutrophils. Possibly, bacteria utilizes neutrophil-derived NETs as a scaffold for released biofilms. In agreement with this, in vivo treatment with ROS-scavengers, NETs disruption or usage of the bacterial strains unable to bind DNA, suppress neutrophil-mediated biofilm formation in the lungs. Together, our findings indicate that the excessive activation of neutrophils by type I IFNs leads to their boosted NETosis that in turn triggers biofilm formation by P. aeruginosa and supports its persistence in the infected lung. Targeting these mechanisms could offer a new therapeutic approach to prevent persistent bacterial infections in patients with diseases associated with the up-regulation of type I IFNs.
    • Evolutionary conservation of essential and highly expressed genes in Pseudomonas aeruginosa.

      Dötsch, Andreas; Klawonn, Frank; Jarek, Michael; Scharfe, Maren; Blöcker, Helmut; Häussler, Susanne; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2010)
      The constant increase in development and spread of bacterial resistance to antibiotics poses a serious threat to human health. New sequencing technologies are now on the horizon that will yield massive increases in our capacity for DNA sequencing and will revolutionize the drug discovery process. Since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus.
    • Genetic determinants of Pseudomonas aeruginosa fitness during biofilm growth.

      Schinner, Silvia; Engelhardt, Florian; Preusse, Matthias; Thöming, Janne Gesine; Tomasch, Jürgen; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-04-02)
      Pseudomonas aeruginosa is an environmental bacterium and an opportunistic human pathogen. It is also a well-established model organism to study bacterial adaptation to stressful conditions, such as those encountered during an infection process in the human host. Advancing knowledge on P. aeruginosa adaptation to biofilm growth conditions is bound to reveal novel strategies and targets for the treatment of chronic biofilm-associated infections. Here, we generated transposon insertion libraries in three P. aeruginosa strain backgrounds and determined the relative frequency of each insertion following biofilm growth using transposon sequencing. We demonstrate that in general the SOS response, several tRNA modifying enzymes as well as adaptation to microaerophilic growth conditions play a key role in bacterial survival under biofilm growth conditions. On the other hand, presence of genes involved in motility and PQS signaling were less important during biofilm growth. Several mutants exhibiting transposon insertions in genes detected in our screen were validated for their biofilm growth capabilities and biofilm specific transcriptional responses using independently generated transposon mutants. Our results provide new insights into P. aeruginosa adaptation to biofilm growth conditions. The detection of previously unknown determinants of biofilm survival supports the use of transposon insertion sequencing as a global genomic technology for understanding the establishment of difficult to treat biofilm-associated infections.
    • Host-induced spermidine production in motile triggers phagocytic uptake.

      Felgner, Sebastian; Preusse, Matthias; Beutling, Ulrike; Stahnke, Stephanie; Pawar, Vinay; Rohde, Manfred; Brönstrup, Mark; Stradal, Theresia; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (elifeSciences, 2020-09-22)
      Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.
    • Identification and quantification of (t)RNA modifications in Pseudomonas aeruginosa by liquid chromatography-tandem mass spectrometry.

      Grobe, Svenja; Doberenz, Sebastian; Ferreira, Kevin; Krueger, Jonas; Brönstrup, Mark; Kaever, Volkhard; Häußler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2019-01-15)
      Transfer RNA (tRNA) modifications impact the structure and function of tRNAs thus affecting the efficiency and fidelity of translation. In the opportunistic pathogen Pseudomonas aeruginosa translational regulation plays an important but less defined role in the adaptation to changing environments. In this study, we explored tRNA modifications in P. aeruginosa using LC-MS/MS based approaches. Neutral Loss Scan (NLS) demonstrated the potential to identify previously unknown modifications, while Multiple Reaction Monitoring (MRM) can detect modifications with high specificity and sensitivity. In this study, the MRM-based external calibration method allowed for quantification of the 4 canonical and 32 modified ribonucleosides, of which 21 tRNA modifications were quantified in the total tRNA pool of P. aeruginosa PA14. We also purified the single tRNA isoacceptors tRNA-ArgUCU, tRNA-LeuCAA and tRNA-TrpCCA and determined, both qualitatively and quantitatively, their specific modification pattern. Deeper insights into the nature and dynamics of tRNA modifications in P. aeruginosa will pave the way for further studies on posttranscriptional gene regulation as a relatively unexplored molecular mechanism of controlling bacterial pathogenicity and life style.
    • Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity.

      Blanka, Andrea; Schulz, Sebastian; Eckweiler, Denitsa; Franke, Raimo; Bielecka, Agata; Nicolai, Tanja; Casilag, Fiordiligie; Düvel, Juliane; Abraham, Wolf-Rainer; Kaever, Volkhard; et al. (2014-01)
      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.
    • Organism-specific depletion of highly abundant RNA species from bacterial total RNA.

      Engelhardt, Florian; Tomasch, Jürgen; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Microbiology Society, 2020-09-09)
      High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.
    • The Peptide Chain Release Factor Methyltransferase PrmC Influences the Pseudomonas aeruginosa PA14 Endo- and Exometabolome.

      Depke, Tobias; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-18)
      Pseudomonas aeruginosa is one of the most important nosocomial pathogens and understanding its virulence is the key to effective control of P. aeruginosa infections. The regulatory network governing virulence factor production in P. aeruginosa is exceptionally complex. Previous studies have shown that the peptide chain release factor methyltransferase PrmC plays an important role in bacterial pathogenicity. Yet, the underlying molecular mechanism is incompletely understood. In this study, we used untargeted liquid and gas chromatography coupled to mass spectrometry to characterise the metabolome of a prmC defective P. aeruginosa PA14 strain in comparison with the corresponding strain complemented with prmC in trans. The comprehensive metabolomics data provided new insight into the influence of prmC on virulence and metabolism. prmC deficiency had broad effects on the endo- and exometabolome of P. aeruginosa PA14, with a marked decrease of the levels of aromatic compounds accompanied by reduced precursor supply from the shikimate pathway. Furthermore, a pronounced decrease of phenazine production was observed as well as lower abundance of alkylquinolones. Unexpectedly, the metabolomics data showed no prmC-dependent effect on rhamnolipid production and an increase in pyochelin levels. A putative virulence biomarker identified in a previous study was significantly less abundant in the prmC deficient strain.
    • The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing.

      Dötsch, Andreas; Eckweiler, Denitsa; Schniederjans, Monika; Zimmermann, Ariane; Jensen, Vanessa; Scharfe, Maren; Geffers, Robert; Häussler, Susanne; Helmholtz Centre of infection research; Inhoffenstr. 7; D-38124 Braunschweig; Germany. (2012)
      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.
    • Targeting Bacterial Gyrase with Cystobactamid, Fluoroquinolone, and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa.

      Franke, Raimo; Overwin, Heike; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2021-07-13)
      The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins, and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (eDNA) release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account. IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.
    • Untargeted LC-MS Metabolomics Differentiates Between Virulent and Avirulent Clinical Strains of Pseudomonas aeruginosa

      Depke, Tobias; Thöming, Janne Gesine; Kordes, Adrian; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-13)
      Pseudomonas aeruginosa is a facultative pathogen that can cause, inter alia, acute or chronic pneumonia in predisposed individuals. The gram-negative bacterium displays considerable genomic and phenotypic diversity that is also shaped by small molecule secondary metabolites. The discrimination of virulence phenotypes is highly relevant to the diagnosis and prognosis of P. aeruginosa infections. In order to discover small molecule metabolites that distinguish different virulence phenotypes of P. aeruginosa, 35 clinical strains were cultivated under standard conditions, characterized in terms of virulence and biofilm phenotype, and their metabolomes were investigated by untargeted liquid chromatography-mass spectrometry. The data was both mined for individual candidate markers as well as used to construct statistical models to infer the virulence phenotype from metabolomics data. We found that clinical strains that differed in their virulence and biofilm phenotype also had pronounced divergence in their metabolomes, as underlined by 332 features that were significantly differentially abundant with fold changes greater than 1.5 in both directions. Important virulence-associated secondary metabolites like rhamnolipids, alkyl quinolones or phenazines were found to be strongly upregulated in virulent strains. In contrast, we observed little change in primary metabolism. A hitherto novel cationic metabolite with a sum formula of C12H15N2 could be identified as a candidate biomarker. A random forest model was able to classify strains according to their virulence and biofilm phenotype with an area under the Receiver Operation Characteristics curve of 0.84. These findings demonstrate that untargeted metabolomics is a valuable tool to characterize P. aeruginosa virulence, and to explore interrelations between clinically important phenotypic traits and the bacterial metabolome.