Helge Karch

Publications of Helge Karch

• The human H3N2 influenza viruses A/Victoria/3/75 and A/Hiroshima/52/2005 preferentially bind to α2,3-sialylated monosialogangliosides with fucosylated poly-N-acetyllactosaminyl chains.

  Authors: Iris Meisen, Tabea Dzudzek, Christina Ehrhardt, Stephan Ludwig, Michael Mormann, Regina Rosenbrück, Regine Lümen, Bernhard Kniep, Helge Karch, Johannes Müthing

  Glycobiology. 04/2012;

  Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annual half million deaths worldwide. Influenza A virusAmong influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annual half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed in thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewis(x) (sLe(x)) monoclonal antibodies showed identical binding patterns compared to those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLe(x)-gangliosides with terminal Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc-epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLe(x)-gangliosides with nLc8-, nLc10- and nLc12Cer-cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses.

• Identification of Intermediate in Evolutionary Model of Enterohemorrhagic Escherichia coli O157.

  Authors: Christian Jenke, Shana R Leopold, Thomas Weniger, Jörg Rothgänger, Dag Harmsen, Helge Karch, Alexander Mellmann

  Emerging infectious diseases. 04/2012; 18(4):582-8.

  Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionaryHighly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H(-) consists of a stepwise evolution scenario proceeding from O55:H7 to a node (hypothetical intermediate) that then branches into sorbitol-fermenting (SF) O157:H(-) and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H(-) isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H(-). The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.

• Need for Long-term Follow-up in Enterohemorrhagic Escherichia coli-Associated Hemolytic Uremic Syndrome Due to Late-Emerging Sequelae.

  Authors: Alejandra Rosales, Johannes Hofer, Lothar-Bernd Zimmerhackl, Therese C Jungraithmayr, Magdalena Riedl, Thomas Giner, Alexander Strasak, Dorothea Orth-Höller, Reinhard Würzner, Helge Karch

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 03/2012;

  Background. The aim of this study was to evaluate the long-term prognosis of children with hemolytic uremic syndrome (HUS).Methods. Over a 6-year period, 619 pediatric patients with the clinicalBackground. The aim of this study was to evaluate the long-term prognosis of children with hemolytic uremic syndrome (HUS).Methods. Over a 6-year period, 619 pediatric patients with the clinical diagnosis of HUS were registered in Austria and Germany, and a subset (n = 274) was prospectively followed up for 5 years.Results. Infection with enterohemorrhagic Escherichia coli (EHEC) was confirmed in 79% of cases. Five years after diagnosis, 70% of EHEC-infected patients (95% confidence interval [CI], .63-.76) were fully recovered. The remaining 30% had persistent hypertension (9%), neurological symptoms (4%), decreased glomerular filtration rate (7%), and/or proteinuria (18%). Hypertension and proteinuria developed in a total of 18% of patients who had no sequelae 1 year after the acute phase (95% CI, 12-26). Multivariate logistic regression analysis demonstrated an association between the use of plasma therapy during acute phase and poor long-term outcome (odds ratio, 2.9-13; 95% CI, 2.4-33; P < .05), but this treatment was also used more frequently in severe cases. In contrast, the use of antibiotic therapy in the diarrheal phase and other established risk factors for developing HUS, such as Shiga toxin 2 and EHEC serotypes traditionally considered to be "high risk," were not associated with adverse long-term outcome. In particular, there was no difference between O157 and non-O157 EHEC.Conclusions. This study identified an association between the use of plasma treatment and poor long-term outcome and confirms already known risk factors for poor prognosis. Follow-up investigations for at least 5 years are recommended to detect late-emerging sequelae.

• Epidemic Escherichia coli O104:H4: Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction.

  Authors: Martina Bielaszewska, Evgeny A Idelevich, Wenlan Zhang, Andreas Bauwens, Frieder Schaumburg, Alexander Mellmann, Georg Peters, Helge Karch

  Antimicrobial agents and chemotherapy. 03/2012;

  The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing ShigaThe role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated in some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx(2)-harboring bacteriophages, stx(2) transcription and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased (P < 0.001 to P < 0.05), fosfomycin, gentamicin, and kanamycin insignificantly influenced (P > 0.1), and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased (P ≤ 0.05) stx(2)-phage induction and Stx2 production in outbreak isolates. Ciprofloxacin and chloramphenicol significantly upregulated and downregulated, respectively, stx(2) transcription (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which had been used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induce stx(2)-harboring phages nor increase stx(2) transcription and Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options in patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

• Real-time multiplex polymerase chain reaction for detecting Shiga toxin 2-producing Escherichia coli O104:H4 in human stools.

  Authors: Wenlan Zhang, Martina Bielaszewska, Andreas Bauwens, Angelika Fruth, Alexander Mellmann, Helge Karch

  Journal of clinical microbiology. 02/2012;

  A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHECA real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and 132 patients' stool enrichment cultures. It is a rapid, sensitive and specific tool for detecting EHEC O104:H4 in human stools.

• Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.

  Authors: Johannes Müthing, Iris Meisen, Wenlan Zhang, Martina Bielaszewska, Michael Mormann, Rolf Bauerfeind, M Alexander Schmidt, Alexander W Friedrich, Helge Karch

  Glycobiology. 01/2012;

  Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurologicalShiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders, and often fatal outcome. Stx2e-producing strains from the intestine of slaughtered pigs (n=3), feces of piglets with postweaning diarrhea or edema disease (n=12) and feces of humans with asymptomatic infections or mild diarrhea (n=13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.

• Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104:H4 Outbreak Strains.

  Authors: Leighton Pritchard, Nicola J Holden, Martina Bielaszewska, Helge Karch, Ian K Toth

  PloS one. 01/2012; 7(4):e34498.

  An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreakAn Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates. Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 'positive' E. coli O104:H4 outbreak and 32 'negative' non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity. Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.

• Comparison of multilocus variable-number tandem-repeat analysis and multilocus sequence typing for differentiation of hemolytic-uremic syndrome-associated Escherichia coli (HUSEC) collection strains.

  Authors: Christian Jenke, Björn Arne Lindstedt, Dag Harmsen, Helge Karch, Lin Thorstensen Brandal, Alexander Mellmann

  Journal of clinical microbiology. 08/2011; 49(10):3644-6.

  Multilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coliMultilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli strains. Although MLVA--like MLST--was highly discriminatory (index of diversity, 0.988 versus 0.984), a low level of concordance demonstrated the limited ability of MLVA to reflect long-term evolutionary events.

• Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study.

  Authors: Martina Bielaszewska, Alexander Mellmann, Wenlan Zhang, Robin Köck, Angelika Fruth, Andreas Bauwens, Georg Peters, Helge Karch

  The Lancet infectious diseases. 06/2011; 11(9):671-6.

  In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain (rfb(O104), fliC(H4), stx(2), and terD). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli. We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx(2), iha, lpf(O26), lpf(O113)) and enteroaggregative E coli (aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. German Federal Ministry of Education and Research, Network Zoonoses.

• Characterization of Escherichia coli strains isolated from patients with diarrhea in Sao Paulo, Brazil: identification of intermediate virulence factor profiles by multiplex PCR.

  Authors: Ariane Liebchen, Inga Benz, Alexander Mellmann, Helge Karch, Tânia A T Gomes, Denise Yamamoto, Rodrigo T Hernandes, Jorge Sampaio, Suely C F Sampaio, Angelika Fruth, M Alexander Schmidt

  Journal of clinical microbiology. 06/2011; 49(6):2274-8.

  Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients withIntestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (EAEC) strains, followed by atypical EPEC (ATEC) strains. Classical enteropathogenic E. coli (EPEC) strains were not detected.

• Cellular prion proteins in human platelets show a phenotype different to those in brain tissues.

  Authors: Thorsten Kuczius, Julia Kleinert, Helge Karch, Walter Sibrowski, Reinhard Kelsch

  Journal of cellular biochemistry. 03/2011; 112(3):954-62.

  Prion diseases are characterized by high accumulation of infectious prion proteins (PrP(Sc)) in brains. PrP(Sc) are propagated by the conversion of host-encoded cellular prion proteins (PrP(C)) whichPrion diseases are characterized by high accumulation of infectious prion proteins (PrP(Sc)) in brains. PrP(Sc) are propagated by the conversion of host-encoded cellular prion proteins (PrP(C)) which are essential for developing the disease but are heterogeneously expressed in brains. The disease can be transmitted to humans and animals through blood and blood products, however, little attention has been given to molecular characterization of PrP(C) in blood cells. In this presented study, we characterized phenotypically PrP(C) of platelets (plt) and characterized the proteins regarding their glycobanding profiles by quantitative immunoblotting using a panel of monoclonal antibodies. The glycosylation patterns of plt and brain PrP(C) were compared using the ratios of di-, mono-, and non-glycosylated prions. The detergent solubility of plt and brain PrP(C) was also analyzed. The distinct banding patterns and detergent solubility of plt PrP(C) differed clearly from the glycosylation profiles and solubility characteristics of brain PrP(C). Plt PrP(C) exhibited single or only few prion protein types, whereas brain PrP(C) showed more extensive banding patterns and lower detergent solubility. Plt PrP(C) are post-translational modified differently from PrP(C) in brain. These findings suggest other or less physiological functions of plt PrP(C) than in brain.

• Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype.

  Authors: Martina Bielaszewska, Barbara Middendorf, Phillip I Tarr, Wenlan Zhang, Rita Prager, Thomas Aldick, Ulrich Dobrindt, Helge Karch, Alexander Mellmann

  Molecular microbiology. 02/2011; 79(4):1024-44.

  Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml⁻¹) sustained large internal deletions within the Tel(R) OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10⁻³ and 1.97 × 10⁻⁴ cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10⁻¹ cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting Tel(R) OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.

• Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.

  Authors: Jens Brockmeyer, Thomas Aldick, Jens Soltwisch, Wenlan Zhang, Philip I Tarr, André Weiss, Klaus Dreisewerd, Johannes Müthing, Martina Bielaszewska, Helge Karch

  Environmental microbiology. 02/2011; 13(5):1327-41.

  The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factorsThe haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified EspPα was added to a growing culture of an EHEC-Hly-expressing strain, with isolated proteins and with coexpressing strains, and was independent of the EHEC serotype. EHEC-Hly breakdown occurred as a multistage process with the formation of characteristic fragments with relative molecular masses of ~82 kDa and/or ~84 kDa and ~34 kDa. The initial cleavage occurred in the N-terminal hydrophobic domain of EHEC-Hly between Leu(235) and Ser(236) and abolished its haemolytic activity. In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed. EHEC in contact with human intestinal epithelial cells simultaneously upregulated their EHEC-Hly and EspP indicating that both molecules might interact under physiological conditions. We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors. Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.

• Watch out for the even eviler cousin-sorbitol-fermenting E coli O157.

  Authors: Dirk Werber, Martina Bielaszewska, Christina Frank, Klaus Stark, Helge Karch

  Lancet. 01/2011; 377(9762):298-9.

• Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.

  Authors: Josefine Betz, Martina Bielaszewska, Andrea Thies, Hans-Ulrich Humpf, Klaus Dreisewerd, Helge Karch, Kwang S Kim, Alexander W Friedrich, Johannes Müthing

  Journal of lipid research. 01/2011; 52(4):618-34.

  Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majorityVascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.

• Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.

  Authors: Alexander Mellmann, Dag Harmsen, Craig A Cummings, Emily B Zentz, Shana R Leopold, Alain Rico, Karola Prior, Rafael Szczepanowski, Yongmei Ji, Wenlan Zhang, Stephen F McLaughlin, John K Henkhaus, Benjamin Leopold, Martina Bielaszewska, Rita Prager, Pius M Brzoska, Richard L Moore, Simone Guenther, Jonathan M Rothberg, Helge Karch

  PloS one. 01/2011; 6(7):e22751.

  An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deathsAn ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

• Normal silica gel and reversed phase thin-layer chromatography coupled with UV spectroscopy and IR-MALDI-o-TOF-MS for the detection of tetracycline antibiotics.

  Authors: Iris Meisen, Sebastian Wisholzer, Jens Soltwisch, Klaus Dreisewerd, Michael Mormann, Johannes Müthing, Helge Karch, Alexander W Friedrich

  Analytical and bioanalytical chemistry. 12/2010; 398(7-8):2821-31.

  Tetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in humanTetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in human and veterinary medicine. Here, we introduce the combination of UV spectroscopic detection of high-performance thin-layer chromatography (HPTLC)-separated TCs with direct analysis on solid phase using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). Normal silica gel phase- and water-wettable hybrid C(18) reversed phase layers (RP-18W) both allowed HPTLC separation and sensitive UV spectroscopic detection followed by MS analysis of distinct TCs bands using the liquid matrix glycerol. The novel approach of direct IR-MALDI-o-TOF-MS analysis resulted in the unequivocal identification of four structurally different TCs employed in this study, and linear calibration curves were produced for analyte amounts from 20 ng to 1 μg. MS analysis of TCs from RP-18W HPTLC plates was found to be superior when compared to the spectra acquired from the silica gel layer. Ionic analytes obtained from the RP-18W surface are mainly detected as protonated species of high abundance accompanied by a reduced formation of adducts as well as background ions arising from the matrix or the stationary phase. This results in decreased complexity of the spectra and enhanced sensitivity of the combinatorial method. An approximate detection limit of 5 ng of individual TCs in mixtures by combining RP-18W HPTLC with IR-MALDI-o-TOF-MS offers a novel timely and cost-efficient method for tracing TCs.

• Subtyping of human cellular prion proteins and their differential solubility.

  Authors: Thorsten Kuczius, Janet Wohlers, Helge Karch, Martin H Groschup

  Experimental neurology. 11/2010; 227(1):188-94.

  A human form of a prion disorder is the Creutzfeldt-Jakob disease. A hallmark of the disease is the accumulation of misfolded prion proteins (PrP(Sc)), which exist as heterogeneous subtypes. PrP(Sc)A human form of a prion disorder is the Creutzfeldt-Jakob disease. A hallmark of the disease is the accumulation of misfolded prion proteins (PrP(Sc)), which exist as heterogeneous subtypes. PrP(Sc) is formed by protein conversion from the host-encoded cellular prion (PrP(C)), which is expressed and modified to various isoforms. Little is known about variation in PrP(C); however, it is assumed that PrP(C) types play important roles in the formation of PrP(Sc). In this study, we separated distinct human PrP(C) subtypes on the basis of differential protein solubilities in detergent solutions. Single and sequential application of the detergents Triton X-100, octyl-glucopyranoside and CHAPS facilitated high solubility of glycosylated PrP(C) isoforms, whereas high proportions of nonglycosylated PrP(C) remained non-soluble. Most proteins became highly soluble with laurylsarcosine and sodium dodecyl sulphate. Our findings demonstrate that the solubility characteristics of heterogeneous PrP(C) overlap in human brains and convey distinct solubility subtypes. Differentiation by solubility experiments can therefore provide valuable information on prion protein composition, facilitate the separation of subtypes, and offer new prospects for conversion specificity of distinct isoforms.

• Immunological one-step determination of the central nervous system indicator proteins, neuron-specific enolase and glial fibrillary acidic protein, in meat products.

  Authors: Thorsten Kuczius, Olga Böhler, Helge Karch

  Molecular nutrition & food research. 11/2010; 54(11):1690-5.

  The determination of specific marker proteins is important in the prevention of infections and transmission of disease. Several diagnostic assays have been developed but these are mostly restrictedThe determination of specific marker proteins is important in the prevention of infections and transmission of disease. Several diagnostic assays have been developed but these are mostly restricted to the detection of single antigens. Thus there is a need for multiplex detection assays for the simultaneous analysis of several disease indicators. Consumer protection against the transmission of prion diseases is ensured by the removal of specified risk material from cattle meat during slaughtering and this is regulated by law. The investigation reported here describes a one-step determination on immunoblots of the simultaneous detection of two indicator proteins, neuron-specific enolase and glial fibrillary acidic protein, in tissues of the central nervous system. Although the two proteins run in polyacrylamide gels with similar molecular masses the indicators are differentiated by immunological reactions followed by visualizing the different coloured specific protein bands which develop. The enolase exhibits a brown band, whereas the glial fibrillary acidic protein is red. Immunoblotting has proved to be a suitable assay for multiplex analysis of marker proteins possessing similar molecular weights and is therefore a suitable tool for application in food, veterinary and medical facilities.

• A duplex approach for immunochemical staining and typing of proteins in Western blots.

  Authors: Thorsten Kuczius, Lars Brandstädter, Helge Karch, Jan P M Langeveld

  Analytical biochemistry. 10/2010; 409(2):260-6.

  The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. ProteinThe qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a specific banding pattern. For protein characterization, several antibodies that recognize different epitopes within the protein sequence are used. However, repeated or parallel gel runs are needed. Here we describe a sequential determination of prion proteins in healthy and pathological states that both consist of di-, mono-, and nonglycosylated isoforms using a single blot with two antibodies from two species that recognize one antigen with two epitopes. The band signals are visualized by using different chemiluminescent substrate reactions. This application can be used in the fields of diagnostics and public health to detect full-length and fragmented proteins and can also be used for characterization of overlaying proteins.