• Unravelling post-transcriptional PrmC-dependent regulatory mechanisms in Pseudomonas aeruginosa.

      Krueger, Jonas; Pohl, Sarah; Preusse, Matthias; Kordes, Adrian; Rugen, Nils; Schniederjans, Monika; Pich, Andreas; Häussler, Susanne; Twincore, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2016-10)
      Transcriptional regulation has a central role in cellular adaptation processes and is well investigated. In contrast, the importance of the post-transcriptional regulation on these processes is less well defined. The technological advancements have been critical to precisely quantify protein and mRNA level changes and hold promise to provide more insights into how post-transcriptional regulation determines phenotypes. In Pseudomonas aeruginosa the methyltransferase PrmC methylates peptide chain release factors to facilitate translation termination. Loss of PrmC activity abolishes anaerobic growth and leads to reduced production of quorum sensing-associated virulence factors. Here, by applying SILAC technology in combination with mRNA-sequencing, they provide evidence that the P. aeruginosa phenotype can be attributed to a change in protein to mRNA ratios of selected protein groups. The UAG-dependent translation termination was more dependent on PrmC activity than the UAA- and UGA-dependent translation termination. Additionally, a bias toward UAG stop codons in global transcriptional regulators was found. The finding that this bias in stop codon usage determines the P. aeruginosa phenotype is unexpected and adds complexity to regulatory circuits. Via modulation of PrmC activity the bacterial cell can cross-regulate targets independently of transcriptional signals, a process with an underestimated impact on the bacterial phenotype.
    • 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.
    • Use of Single-Frequency Impedance Spectroscopy to Characterize the Growth Dynamics of Biofilm Formation in Pseudomonas aeruginosa.

      van Duuren, Jozef B J H; Müsken, Mathias; Karge, Bianka; Tomasch, Jürgen; Wittmann, Christoph; Häussler, Susanne; Brönstrup, Mark (2017-07-12)
      Impedance spectroscopy has been applied in prokaryotic and eukaryotic cytometry as a label-free method for the investigation of adherent cells. In this paper, its use for characterizing the growth dynamics of P. aeruginosa biofilms is described and compared to crystal violet staining and confocal microscopy. The method allows monitoring the growth of biofilm-forming P. aeruginosa in a continuous and label-free manner over a period of 72 h in a 96 well plate format. Impedance curves obtained for P. aeruginosa PA14 wild type and mutant strains with a transposon insertion in pqsA and pelA genes exhibited distinct phases. We propose that the slope of the declining curve following a maximum at ca. 35-40 h is a measure of biofilm formation. Transplant experiments with P. aeruginosa biofilms and paraffin suggest that the impedance also reflects pellicle formation at the liquid-air interface, a barely considered contributor to impedance. Finally, the impairment of biofilm formation upon treatment of cultures with L-arginine and with ciprofloxacin, tobramycin and meropenem was studied by single frequency impedance spectroscopy. We suggest that these findings qualify impedance spectroscopy as an additional technique to characterize biofilm formation and its modulation by small molecule drugs.
    • Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis.

      Kayser, Hartmut; Nimtz, Manfred; Ringler, Philippe; Müller, Shirley A; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2016-01)
      Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.
    • The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways.

      Malone, Jacob G; Jaeger, Tina; Manfredi, Pablo; Dötsch, Andreas; Blanka, Andrea; Bos, Raphael; Cornelis, Guy R; Häussler, Susanne; Jenal, Urs; Biozentrum of the University of Basel, Basel, Switzerland. (2012-06)
      The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.