• The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

      Solbak, Sara M; Wray, Victor; Horvli, Ole; Raae, Arnt J; Flydal, Marte I; Henklein, Petra; Henklein, Peter; Nimtz, Manfred; Schubert, Ulrich; Fossen, Torgils (2011-12-20)
      Abstract Background Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear. Results Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA. Conclusions For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.
    • Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

      Vanz, Ana L; Lünsdorf, Heinrich; Adnan, Ahmad; Nimtz, Manfred; Gurramkonda, Chandrasekhar; Khanna, Navin; Rinas, Ursula (2012-08-08)
      Abstract Background Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. Conclusions In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.
    • Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection

      Worthmann, Hans; Tryc, Anita B; Dirks, Meike; Schuppner, Ramona; Brand, Korbinian; Klawonn, Frank; Lichtinghagen, Ralf; Weissenborn, Karin (2015-01-23)
      Abstract Background Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection. Methods Fifty-six patients with ischemic stroke (n = 51) and transient ischemic attack (TIA) (n = 5) who presented within 6 hours after symptom onset and who were free of detectable infection on admission were included in the study. Of these, 20 developed early infections during the first week. Blood samples were taken at 6, 12, and 24 hours and at 3 and 7 days after stroke onset. Levels of LBP, Il-10, IL-6 and CRP, as well as S100B, were measured as markers of inflammation and brain damage by commercially available immunometric tests. Results In the univariate analysis, levels of LBP, IL-10, IL-6 and CRP significantly differed between patients who developed an infection and those who did not. In the binary logistic regression analysis, which was adjusted for National Institutes of Health Stroke Scale (NIHSS) on admission, stroke subtype and S100B peak levels, as indicator of the extent of brain damage, IL-10 at 6 hours, CRP at 6 hours and NIHSS on admission were identified as independent predictors of infection (IL-10: P = 0.009; CRP: P = 0.018; NIHSS: P = 0.041). The area under the curve (AUC) of the receiver operating characteristic (ROC) curves in relation to the dichotomized status of the infection (infection versus no infection) was 0.74 (95% confidence interval: 0.59 to 0.88) for CRP at 6 hours, 0.76 (0.61 to 0.9) for IL-10 at 6 hours, 0.83 (0.71 to 0.94) for NIHSS on admission and 0.94 (0.88 to 1) for the combination of CRP, IL-10 and NIHSS. In a subanalysis, 16 patients with early infections were matched with 16 patients without infection according to S100B peak levels. Here, the temporal pattern of LBP, IL-10, IL-6 and CRP significantly differed between the patient groups. Conclusions Our data show that blood levels of inflammation markers may be used as early predictors of stroke-associated infection. We propose prospective studies to investigate if the calculated cut-offs of CRP, IL-10 and NIHSS might help to identify patients who should receive early preventive antibiotic treatment.
    • Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli

      Coldewey, Sina M; Hartmann, Maike; Schmidt, Dorothea S; Engelking, Uta; Ukena, Sya N; Gunzer, Florian (2007-03-26)
      Abstract Background Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. Methods Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. Results First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. Conclusion The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.
    • Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation

      Della Beffa, Cristina; Klawonn, Frank; Menetski, Joseph P; Schumacher, H R; Pessler, Frank (2011-10-25)
      Abstract Background The murine air pouch membrane represents an easily accessible tissue for studies on gene regulation in acute inflammation. Considering that acute inflammation may affect expression of molecular reference genes, we evaluated the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prolylpeptidyl isomerase A (PPIA) in the air pouch membrane during a complete time course of urate crystal inflammation and correlated the results with expression of interleukin (IL)-1β and hypoxia inducible factor (HIF)-1α. In addition, we aimed to identify alternate potential reference genes. Methods Using custom microfluidic real-time PCR arrays, the expression of 96 genes including GAPDH, PPIA, IL-1β, and HIF-1α was determined in dissected air pouch membranes 1, 4, 9, 18, 27, and 50 hours (h) after injecting monosodium urate (MSU) crystals into the pouch. One-way ANOVA was used to detect differential gene expression throughout the time course. Using the genes on these arrays as a convenience sample, alternate candidate reference genes were sought (1) with a biostatistical approach and (2) using the geNorm software tool. Results Pouch leukocytes peaked at t = 9h and declined toward t = 50h. PPIA expression was not differentially regulated (p = 0.52, ANOVA). In contrast, GAPDH mRNA increased steadily after crystal injection, reaching a maximal 2.8-fold increase at t = 18h (p = 0.0006, t test), which followed a marked induction of IL-1β (max., 208-fold at t = 4h, p = 8.4 × 10-5, t test) and HIF-1α (max., 6.6-fold at t = 4h, p = 0.00025, t test). Fifteen genes were artifactually identified as "significantly regulated" when Ct values were normalized against GAPDH expression. The biostatistical approach and the geNorm analysis identified overlapping sets of candidate reference genes. Both ranked PPIA as the best candidate, followed by defender against cell death 1 (DAD1) and high-mobility group B1 (HMGB1). Conclusions GAPDH mRNA expression is up-regulated in urate crystal inflammation, possibly due to inflammation-associated hypoxia. Using GAPDH mRNA for molecular normalization resulted in significant artifacts in the calculated expression of the target mRNAs. PPIA and other stably expressed genes promise to be more appropriate reference genes in this model.
    • Infection- and procedure-dependent effects on pulmonary gene expression in the early phase of influenza A virus infection in mice

      Preusse, Matthias; Tantawy, Mohamed A; Klawonn, Frank; Schughart, Klaus; Pessler, Frank (2013-12-17)
      Abstract Background Investigating the host response in the early stage of influenza A virus (IAV) infection is of considerable interest. However, it is conceivable that effects due to the anesthesia and/or intranasal infection procedure might introduce artifacts. We therefore aimed to evaluate the effects of anesthesia and/or intranasal infection on transcription of selected pulmonary mRNAs in two inbred mouse strains with differential susceptibility to IAV infection. Results DBA/2J and C57BL/6J mice were evaluated in a time course experiment in which lung tissue was sampled after 6, 12, 18, 24, 48 and 120 h. After anesthesia with ketamine and xylazine, a suspension of mouse-adapted IAV strain PR8_Mun in 20 μl sterile buffer, or 20 μl sterile buffer only, was instilled intranasally. The mice receiving anesthesia and PBS only were designated the “mock treatment” group. Pulmonary expression of 10 host mRNAs (Fos, Retnla, Irg1, Il6, Il1b, Cxcl10, Stat1, Ifng, Ifnl2, and Mx1) and viral hemagglutinin (HA) mRNA were determined at the designated time points. As expected, weight loss and viral replication were greater in the DBA/2J strain (which is more susceptible to IAV infection). Four mRNAs (Retnla, Irg1, Il6, and Cxcl10) were procedure-dependently regulated in DBA/2J mice between 6 and 24 h, and two (Retnla and Il6) in C57BL/6J mice, although to a lesser extent. All 10 mRNAs rose after infection, but one (Fos) only in DBA/2J mice. These infection-dependent effects could be separated from procedure-dependent effects beginning around 12 h in DBA/2J and 18 h in C57BL/6J mice. The interferon-related mRNAs Stat1, Ifng, Infl2, and Mx1 were unaffected by mock treatment in either mouse strain. Mx1 and Infl2 correlated best with HA mRNA expression (r = 0.97 and 0.93, respectively, in DBA/2J). Conclusions These results demonstrate effects of the anesthesia and/or intranasal infection procedure on pulmonary gene expression, which are detectable between approximately 6 and 24 h post procedure and vary in intensity and temporal evolution depending on the mouse strain used. Mock infection controls should be included in all studies on pulmonary gene expression in the early phase of infection with IAV and, likely, other respiratory pathogens.
    • Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes

      Hain, Torsten; Ghai, Rohit; Billion, André; Kuenne, Carsten T; Steinweg, Christiane; Izar, Benjamin; Mohamed, Walid; Mraheil, Mobarak A; Domann, Eugen; Schaffrath, Silke; Kärst, Uwe; Goesmann, Alexander; Oehm, Sebastian; Pühler, Alfred; Merkl, Rainer; Vorwerk, Sonja; Glaser, Philippe; Garrido, Patricia; Rusniok, Christophe; Buchrieser, Carmen; Goebel, Werner; Chakraborty, Trinad (2012-04-24)
      Abstract Background Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. Results The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. Conclusion Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.
    • Analysis of Practical Identifiability of a Viral Infection Model.

      Nguyen, Van Kinh; Klawonn, Frank; Mikolajczyk, Rafael; Hernandez-Vargas, Esteban A.; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Mathematical modelling approaches have granted a significant contribution to life sciences and beyond to understand experimental results. However, incomplete and inadequate assessments in parameter estimation practices hamper the parameter reliability, and consequently the insights that ultimately could arise from a mathematical model. To keep the diligent works in modelling biological systems from being mistrusted, potential sources of error must be acknowledged. Employing a popular mathematical model in viral infection research, existing means and practices in parameter estimation are exemplified. Numerical results show that poor experimental data is a main source that can lead to erroneous parameter estimates despite the use of innovative parameter estimation algorithms. Arbitrary choices of initial conditions as well as data asynchrony distort the parameter estimates but are often overlooked in modelling studies. This work stresses the existence of several sources of error buried in reports of modelling biological systems, voicing the need for assessing the sources of error, consolidating efforts in solving the immediate difficulties, and possibly reconsidering the use of mathematical modelling to quantify experimental data.
    • The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

      Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-04-26)
      Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.
    • Diagnostic Support for Selected Paediatric Pulmonary Diseases Using Answer-Pattern Recognition in Questionnaires Based on Combined Data Mining Applications--A Monocentric Observational Pilot Study.

      Rother, Ann-Katrin; Schwerk, Nicolaus; Brinkmann, Folke; Klawonn, Frank; Lechner, Werner; Grigull, Lorenz; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015)
      Clinical symptoms in children with pulmonary diseases are frequently non-specific. Rare diseases such as primary ciliary dyskinesia (PCD), cystic fibrosis (CF) or protracted bacterial bronchitis (PBB) can be easily missed at the general practitioner (GP).
    • Relative ion intensities of maltooligosaccharide ethers in electrospray ionization ion trap mass spectrometry: A quantitative evaluation

      Gangula, Sheetal; Nimtz, Manfred; Mischnick, Petra; Technische Universität Braunschweig, Institute of Food Chemistry, Schleinitzstr. 20, Braunschweig, Germany. (2016-05)
    • Diagnostic support for selected neuromuscular diseases using answer-pattern recognition and data mining techniques: a proof of concept multicenter prospective trial.

      Grigull, Lorenz; Lechner, Werner; Petri, Susanne; Kollewe, Katja; Dengler, Reinhard; Mehmecke, Sandra; Schumacher, Ulrike; Lücke, Thomas; Schneider-Gold, Christiane; Köhler, Cornelia; Güttsches, Anne-Katrin; Kortum, Xiaowei; Klawonn, Frank; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Diagnosis of neuromuscular diseases in primary care is often challenging. Rare diseases such as Pompe disease are easily overlooked by the general practitioner. We therefore aimed to develop a diagnostic support tool using patient-oriented questions and combined data mining algorithms recognizing answer patterns in individuals with selected neuromuscular diseases. A multicenter prospective study for the proof of concept was conducted thereafter.
    • LC/MS Based Monitoring of Endogenous Decay Markers for Quality Assessment of Serum Specimens

      Thumfart, Jörg Oliver; Abidi, Nada; Mindt, Sonani; Costani, Victor; Hofheinz, Ralf; Klawonn, Frank; Findeisen, Peter; 1Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany (2015-05-04)
      Preanalytical variations have major impact on most biological assays. Specifically MS-based multiparametric proteomics analyses of blood specimens are seriously affected by limited protein stability due to high intrinsic proteolytic activity of serum and plasma. However, the direct analysis of sample quality (DASQ) for serum specimens is not readily available. Here we propose the mass spectrometry based monitoring of peptide patterns that are ex vivo changing in a time dependent manner to alleviate these constrains.
    • Fuzzy clustering: More than just fuzzification

      Klawonn, Frank; Kruse, Rudolf; Winkler, Roland; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015-12)
    • Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection.

      Worthmann, Hans; Tryc, Anita B; Dirks, Meike; Schuppner, Ramona; Brand, Korbinian; Klawonn, Frank; Lichtinghagen, Ralf; Weissenborn, Karin; Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30623, Hannover, Germany. (2015)
      Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection.
    • Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

      Shevchuk, Olga; Pägelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel; Steinert, Michael; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-11)
      L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.
    • GeneReporter--sequence-based document retrieval and annotation.

      Bartsch, Annekathrin; Bunk, Boyke; Haddad, Isam; Klein, Johannes; Münch, Richard; Johl, Thorsten; Kärst, Uwe; Jänsch, Lothar; Jahn, Dieter; Retter, Ida; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2011-04-01)
      GeneReporter is a web tool that reports functional information and relevant literature on a protein-coding sequence of interest. Its purpose is to support both manual genome annotation and document retrieval. PubMed references corresponding to a sequence are detected by the extraction of query words from UniProt entries of homologous sequences. Data on protein families, domains, potential cofactors, structure, function, cellular localization, metabolic contribution and corresponding DNA binding sites complement the information on a given gene product of interest.
    • Peptidases released by necrotic cells control CD8+ T cell cross-priming.

      Gamrekelashvili, Jaba; Kapanadze, Tamar; Han, Miaojun; Wissing, Josef; Ma, Chi; Jaensch, Lothar; Manns, Michael P; Armstrong, Todd; Jaffee, Elizabeth; White, Ayla O; Citrin, Deborah E; Korangy, Firouzeh; Greten, Tim F (2013-10-08)
      Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.
    • Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions.

      Tielen, Petra; Rosin, Nathalie; Meyer, Ann-Kathrin; Dohnt, Katrin; Haddad, Isam; Jänsch, Lothar; Klein, Johannes; Narten, Maike; Pommerenke, Claudia; Scheer, Maurice; Schobert, Max; Schomburg, Dietmar; Thielen, Bernhard; Jahn, Dieter; Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany. p.tielen@tu-bs.de (2013)
      Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.
    • Proteome analysis of distinct developmental stages of human natural killer (NK) cells.

      Scheiter, Maxi; Lau, Ulrike; van Ham, Marco; Bulitta, Björn; Gröbe, Lothar; Garritsen, Henk; Klawonn, Frank; König, Sebastian; Jänsch, Lothar; Research Group Cellular Proteomics, Helmholtz Centre for Infection Research, HZI, Inhoffenstraβe 7, D-38124 Braunschweig, Germany. (2013-05)
      The recent Natural Killer (NK) cell maturation model postulates that CD34(+) hematopoietic stem cells (HSC) first develop into CD56(bright) NK cells, then into CD56(dim)CD57(-) and finally into terminally maturated CD56(dim)CD57(+). The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56(bright) NK cells versus CD56(dim) NK cells and CD56(dim)CD57(-) NK cells versus CD56(dim)CD57(+) NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa.