• Pain drawings as a diagnostic tool for the differentiation between two pain-associated rare diseases (Ehlers-Danlos-Syndrome, Guillain-Barré-Syndrome).

      Wester, Larissa; Mücke, Martin; Bender, Tim Theodor Albert; Sellin, Julia; Klawonn, Frank; Conrad, Rupert; Szczypien, Natasza; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2020-11-17)
      Background: The diagnosis of rare diseases poses a particular challenge to clinicians. This study analyzes whether patients' pain drawings (PDs) help in the differentiation of two pain-associated rare diseases, Ehlers-Danlos Syndrome (EDS) and Guillain-Barré Syndrome (GBS). Method: The study was designed as a prospective, observational, single-center study. The sample comprised 60 patients with EDS (3 male, 52 female, 5 without gender information; 39.2 ± 11.4 years) and 32 patients with GBS (10 male, 20 female, 2 without gender information; 50.5 ± 13.7 years). Patients marked areas afflicted by pain on a sketch of a human body with anterior, posterior, and lateral views. PDs were electronically scanned and processed. Each PD was classified based on the Ružička similarity to the EDS and the GBS averaged image (pain profile) in a leave-one-out cross validation approach. A receiver operating characteristic (ROC) curve was plotted. Results: 60-80% of EDS patients marked the vertebral column with the neck and the tailbone and the knee joints as pain areas, 40-50% the shoulder-region, the elbows and the thumb saddle joint. 60-70% of GBS patients marked the dorsal and plantar side of the feet as pain areas, 40-50% the palmar side of the fingertips, the dorsal side of the left palm and the tailbone. 86% of the EDS patients and 96% of the GBS patients were correctly identified by computing the Ružička similarity. The ROC curve yielded an excellent area under the curve value of 0.95.
    • Patient's Experience in Pediatric Primary Immunodeficiency Disorders: Computerized Classification of Questionnaires.

      Mücke, Urs; Klemann, Christian; Baumann, Ulrich; Meyer-Bahlburg, Almut; Kortum, Xiaowei; Klawonn, Frank; Lechner, Werner M; Grigull, Lorenz; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Primary immunodeficiency disorders (PIDs) are a heterogeneous group of more than 200 rare diseases. Timely diagnosis is of uttermost importance. Therefore, we aimed to develop a diagnostic questionnaire with computerized pattern-recognition in order to support physicians to identify suspicious patient histories.
    • Pattern discovery in time series using autoencoder in comparison to nonlearning approaches

      Noering, Fabian Kai Dietrich; Schroeder, Yannik; Jonas, Konstantin; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (IOS Press, 2021-01-01)
      In technical systems the analysis of similar situations is a promising technique to gain information about the system's state, its health or wearing. Very often, situations cannot be defined but need to be discovered as recurrent patterns within time series data of the system under consideration. This paper addresses the assessment of different approaches to discover frequent variable-length patterns in time series. Because of the success of artificial neural networks (NN) in various research fields, a special issue of this work is the applicability of NNs to the problem of pattern discovery in time series. Therefore we applied and adapted a Convolutional Autoencoder and compared it to classical nonlearning approaches based on Dynamic Time Warping, based on time series discretization as well as based on the Matrix Profile. These nonlearning approaches have also been adapted, to fulfill our requirements like the discovery of potentially time scaled patterns from noisy time series. We showed the performance (quality, computing time, effort of parametrization) of those approaches in an extensive test with synthetic data sets. Additionally the transferability to other data sets is tested by using real life vehicle data. We demonstrated the ability of Convolutional Autoencoders to discover patterns in an unsupervised way. Furthermore the tests showed, that the Autoencoder is able to discover patterns with a similar quality like classical nonlearning approaches. © 2021 - IOS Press. All rights reserved.
    • PD-1 Blockade Aggravates Epstein-Barr Virus Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4 T Cell Dysregulations.

      Volk, Valery; Theobald, Sebastian J; Danisch, Simon; Khailaie, Sahamoddin; Kalbarczyk, Maja; Schneider, Andreas; Bialek-Waldmann, Julia; Krönke, Nicole; Deng, Yun; Eiz-Vesper, Britta; et al. (Frontiers, 2021-01-12)
      Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV+ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81+ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34+ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8+ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67+CD30+CD20+EBER+PD-L1+ PTLD with central nervous system (CNS) involvement, mirroring EBV+ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4+ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3+ regulatory CD4+ and CD8+ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8+ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV+ PTLD.
    • 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; et al. (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.
    • 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.
    • Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family.

      Babiychuk, Elena; Vandepoele, Klaas; Wissing, Josef; Garcia-Diaz, Miguel; De Rycke, Riet; Akbari, Hana; Joubès, Jérôme; Beeckman, Tom; Jänsch, Lothar; Frentzen, Margrit; et al. (2011-04-19)
      Plastids are DNA-containing organelles unique to plant cells. In Arabidopsis, one-third of the genes required for embryo development encode plastid-localized proteins. To help understand the role of plastids in embryogenesis and postembryonic development, we characterized proteins of the mitochondrial transcription termination factor (mTERF) family, which in animal models, comprises DNA-binding regulators of mitochondrial transcription. Of 35 Arabidopsis mTERF proteins, 11 are plastid-localized. Genetic complementation shows that at least one plastidic mTERF, BELAYA SMERT' (BSM), is required for embryogenesis. The main postembryonic phenotypes of genetic mosaics with the bsm mutation are severe abnormalities in leaf development. Mutant bsm cells are albino, are compromised in growth, and suffer defects in global plastidic gene expression. The bsm phenotype could be phenocopied by inhibition of plastid translation with spectinomycin. Plastid translation is essential for cell viability in dicotyledonous species such as tobacco but not in monocotyledonous maize. Here, genetic interactions between BSM and the gene encoding plastid homomeric acetyl-CoA carboxylase ACC2 suggest that there is a functional redundancy in malonyl-CoA biosynthesis that permits bsm cell survival in Arabidopsis. Overall, our results indicate that biosynthesis of malonyl-CoA and plastid-derived systemic growth-promoting compounds are the processes that link plant development and plastid gene expression.
    • Pleiotropic Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

      Ünal, Can Murat; Karagöz, Mustafa Safa; Berges, Mareike; Priebe, Christina; Borrero de Acuña, José Manuel; Wissing, Josef; Jänsch, Lothar; Jahn, Dieter; Steinert, Michael; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.
    • A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630Δ.erm

      Troitzsch, Daniel; Zhang, Hao; Dittmann, Silvia; Düsterhöft, Dorothee; Möller, Timon Alexander; Michel, Annika-Marisa; Jänsch, Lothar; Riedel, Katharina; Borrero-de Acuña, José Manuel; Jahn, Dieter; et al. (ASM, 2021-03-03)
      The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.IMPORTANCE The intestinal pathogen Clostridioides difficile is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. C. difficile strain 630Δerm was generated in a laboratory from the patient-isolated strain C. difficile 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. C. difficile researchers working with model strain 630Δerm should be aware of this permanent stress response.
    • 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; et al. (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.
    • Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding

      Quaas, Bastian; Burmeister, Laura; Li, Zhaopeng; NIMTZ, MANFRED; Hoffmann, Andrea; Rinas, Ursula; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
    • Protein Kinase Inhibitors CK59 and CID755673 Alter Primary Human NK Cell Effector Functions.

      Scheiter, Maxi; Bulitta, Björn; van Ham, Marco; Klawonn, Frank; König, Sebastian; Jänsch, Lothar; Research Group Cellular Proteomics, Helmholtz Centre for Infection Research Braunschweig, Germany. (2013)
      Natural killer (NK) cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory therapies. In this study, we tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against calmodulin kinase II (CaMKII; CK59) and PKD family kinases (CID755673) that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Treatment with CK59 and CID755673 indeed resulted in a significant dose-dependent reduction of NK cell degranulation markers and cytokine release in freshly isolated Peripheral blood mononuclear cell populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest protein kinase D2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.
    • Proteogenomic Characterization of the Cement and Adhesive Gland of the Pelagic Gooseneck Barnacle .

      Domínguez-Pérez, Dany; Almeida, Daniela; Wissing, Josef; Machado, André M; Jänsch, Lothar; Antunes, Agostinho; Castro, Luís Filipe; Vasconcelos, Vitor; Campos, Alexandre; Cunha, Isabel; et al. (MDPI, 2021-03-25)
      We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.
    • 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.
    • Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis1

      Kruft, Volker; Eubel, Holger; Jänsch, Lothar; Werhahn, Wolf; Braun, Hans-Peter (American Society of Plant Physiologists, 2001-12)
    • Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

      Wissing, Josef; Jänsch, Lothar; Nimtz, Manfred; Dieterich, Guido; Hornberger, Renate; Kéri, György; Wehland, Jürgen; Daub, Henrik; Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2007-03)
      Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.
    • A publicly accessible database for genome sequences supports tracing of transmission chains and epidemics.

      Frentrup, Martinique; Zhou, Zhemin; Steglich, Matthias; Meier-Kolthoff, Jan P; Göker, Markus; Riedel, Thomas; Bunk, Boyke; Spröer, Cathrin; Overmann, Jörg; Blaschitz, Marion; et al. (Microbiology Society, 2020-07-29)
      Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10-4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile.
    • Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies

      Moreira, Gustavo Marçal Schmidt Garcia; Köllner, Sarah Mara Stella; Helmsing, Saskia; Jänsch, Lothar; Meier, Anja; Gronow, Sabine; Boedeker, Christian; Dübel, Stefan; Mendonça, Marcelo; Moreira, Ângela Nunes; et al. (Springer Science and Business Media LLC, 2020-09-17)
      The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.
    • Quantitative laboratory results: Normal or lognormal distribution?

      Klawonn, Frank; Hoffmann, Georg; Orth, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Walter De Gruyter GmbH, 2020-06-01)
      The identification of a suitable distribution model is a prerequisite for the parametric estimation of reference intervals and other statistical laboratory tasks. Classification of normal vs. lognormal distributions from healthy populations is easy, but from mixed populations, containing unknown proportions of abnormal results, it is challenging. We demonstrate that Bowley’s skewness coefficient differentiates between normal and lognormal distributions. This classifier is robust and easy to calculate from the quartiles Q1–Q3 according to the formula (Q1 − 2 · Q2 + Q3)/(Q3 − Q1). We validate our algorithm with a more complex procedure, which optimizes the exponent λ of a power transformation. As a practical application, we show that Bowley’s skewness coefficient is suited selecting the adequate distribution model for the estimation of reference limits according to a recent International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendation, especially if the data is right-skewed.
    • 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; et al. (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.