• 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.
    • 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.
    • Visualizing production of beta interferon by astrocytes and microglia in brain of La Crosse virus-infected mice.

      Kallfass, Carsten; Ackerman, Andreas; Lienenklaus, Stefan; Weiss, Siegfried; Heimrich, Bernd; Staeheli, Peter; Department of Virology, University of Freiburg, Freiburg, Germany. (2012-10)
      Beta interferon (IFN-β) is a major component of innate immunity in mammals, but information on the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell types responsible for IFN-β production during viral encephalitis, we used reporter mice that express firefly luciferase under the control of the IFN-β promoter and stained organ sections with luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double-staining experiments with cell-type-specific markers revealed that similar numbers of astrocytes and microglia of infected brains were luciferase positive, whereas virus-infected neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast majority of the IFN-β-producing cells in infected brains were astrocytes rather than microglia. Similar conclusions were reached in a second series of experiments in which conditional reporter mice expressing the luciferase reporter gene solely in defined cell types were infected with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than infected neurons represent the major source of IFN-β in LACV-infected mouse brains. They further indicate that IFN-β synthesis in astrocytes and microglia is differentially affected by the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two cell types.
    • Visualizing the beta interferon response in mice during infection with influenza A viruses expressing or lacking nonstructural protein 1.

      Kallfass, Carsten; Lienenklaus, Stefan; Weiss, Siegfried; Staeheli, Peter; Department of Virology, University of Freiburg, Freiburg, Germany. (2013-06)
      The innate host defense against influenza virus is largely dependent on the type I interferon (IFN) system. However, surprisingly little is known about the cellular source of IFN in the infected lung. To clarify this question, we employed a reporter mouse that contains the firefly luciferase gene in place of the IFN-β-coding region. IFN-β-producing cells were identified either by simultaneous immunostaining of lungs for luciferase and cellular markers or by generating conditional reporter mice that express luciferase exclusively in defined cell types. Two different strains of influenza A virus were employed that either do or do not code for nonstructural protein 1 (NS1), which strongly suppresses innate immune responses of infected cells. We found that epithelial cells and lung macrophages, which represent the prime host cells for influenza viruses, showed vigorous IFN-β responses which, however, were severely reduced and delayed if the infecting virus was able to produce NS1. Interestingly, CD11c(+) cell populations that were either expressing or lacking macrophage markers produced the bulk of IFN-β at 48 h after infection with wild-type influenza A virus. Our results demonstrate that the virus-encoded IFN-antagonistic factor NS1 disarms specifically epithelial cells and lung macrophages, which otherwise would serve as main mediators of the early response against infection by influenza virus.
    • 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.