Maike Hartmann

Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam−/− mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam−/− brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam−/− background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam−/− brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Sequence heterogeneity of the echinococcal antigen B

Antigen B is a thermostable lipoprotein of 160 kDa [1] and a major antigen of the hydatid fluid of Echinococcus granulosus metacestodes [2], which is strongly immunogenic in echinococcal disease. About 90% of patients suffering from cystic echinococcosis, caused by the metacestodes of E. granulosus, and 40% of patients suffering from alveolar echinococcosis, caused by Echinococcus multilocularis larvae, exhibited antibodies against antigen B [3]. This suggests that antigen B is expressed in both echinococcal species, although this antigen was previously described as an E. granulosus-specific antigen [4]. In SDS-PAGE, antigen B dissociates and 3 bands of 8, 16 and 24 kDa can be found. Lightowlers et al. [5] observed additional higher-molecular weight molecules, which differed in their masses by 8 kDa, suggesting that antigen

Comparison of a Shiga Toxin Enzyme-Linked Immunosorbent Assay and Two Types of PCR for Detection of Shiga Toxin-Producing Escherichia coli in Human Stool Specimens

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.

Polysialylation of the Synaptic Cell Adhesion Molecule 1 (SynCAM 1) Depends Exclusively on the Polysialyltransferase ST8SiaII in Vivo

Background: Polysialic acid is a developmentally regulated posttranslational modification. Results: Loss of the polysialyltransferase ST8SiaII but not ST8SiaIV abolished polysialylation of SynCAM 1 in the mouse brain. Conclusion: Polysialylation of SynCAM 1 is mediated by ST8SiaII throughout postnatal mouse brain development. Significance: Studying the molecular requirements for protein polysialylation is crucial for understanding how this process is regulated. Polysialic acid is a unique carbohydrate polymer specifically attached to a limited number of glycoproteins. Among them is synaptic cell adhesion molecule 1 (SynCAM 1), a member of the immunoglobulin (Ig) superfamily composed of three extracellular Ig-like domains. Polysialylation of SynCAM 1 is cell type-specific and was exclusively found in NG2 cells, a class of multifunctional progenitor cells that form specialized synapses with neurons. Here, we studied the molecular requirements for SynCAM 1 polysialylation. Analysis of mice lacking one of the two polysialyltransferases, ST8SiaII or ST8SiaIV, revealed that polysialylation of SynCAM 1 is exclusively mediated by ST8SiaII throughout postnatal brain development. Alternative splicing of the three variable exons 8a, 8b, and 8c can theoretically give rise to eight transmembrane isoforms of SynCAM 1. We detected seven transcript variants in the developing mouse brain, including three variants containing exon 8c, which was so far regarded as a cryptic exon in mice. Polysialylation of SynCAM 1 was restricted to four isoforms in perinatal brain. However, cell culture experiments demonstrated that all transmembrane isoforms of SynCAM 1 can be polysialylated by ST8SiaII. Moreover, analysis of domain deletion constructs revealed that Ig1, which harbors the polysialylation site, is not sufficient as an acceptor for ST8SiaII. The minimal polypeptide required for polysialylation contained Ig1 and Ig2, suggesting an important role for Ig2 as a docking site for ST8SiaII.

Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli

BackgroundEnterohemorrhagic 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.MethodsUsing 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.ResultsFirst, 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.ConclusionThe 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.

Identification of a cDNA clone from the larval stage ofEchinococcus granulosus with homologies to theE. multilocularis antigen EM10-expressing cDNA clone

Molecular characteriztion of the tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis in man, resulted, as determined independently by several research groups, in the identification of fulllength or partial cDNA clones with almost identical nucleotide sequences termed EM10 (Frosch et al. 1991), antigen II/3 (Felleisen and Gottstein 1993; Vogel et al. 1988), and EM4 (Hemmings and McManus 1991), respectively. The full-length cDNA of clone EM10 expressed a protein, which was found in the germinal layer of brood capsules and in the tegument of protoscolices (Frosch et al. 1991). This antigen was also most recently described to be expressed by the adult E. muItilocularis worm (Felleisen and Gottstein 1993). Antigen EM10 exhibits a strong homology to proteins of the ERM (ezrin, radixin, moesin) family, the members of which are involved in the linkage of the plasma membrane with the cytoskeleton in eukaryotic cells (Bretscher 1993). EM10 migrates as a 65-kDa band, which is in accordance with the molecular mass predicted from the cDNA sequence. However, a 55-kDa polypeptide is also found when lysates of protoscolices are analyzed by immunoblotting (Frosch et al. 1991). The origin of this smaller protein is not known, but it might be a degradation product. Furthermore, when used for serodiagnosis of echinococcal disease, this echinococcal antigen provides great potential for a specific diagnosis of E. multilocularis infections (Helbig et al. 1993). About 95% of patients suffering from alveolar echinococcosis exhibit antibodies against antigen EM10, but only about 1% of sera from patients suffering from E. granulosus disease elicit an EM10-specific antibody response (Helbig et al. 1993). These observations correlate well with the previ-

9- O -Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate

Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. Supplementary information The online version of this article (doi:10.1038/ncomms8673) contains supplementary material, which is available to authorized users.

Improving health from the inside: Use of engineered intestinal microorganisms as in situ cytokine delivery system

The anti-inflammatory cytokine interleukin-10 and its viral homologs were chosen as model proteins for the development of drug delivery systems based on probiotic carriers like E. coli Nissle 1917, E. coli G3/10, and Saccharomyces boulardii. Exterior cytokine secretion was achieved by a modified E. coli hemolysin transporter. Release of interleukin-10 transported to the periplasm via the OmpF signal peptide was enabled by a T4 phage lysis system under control of the araC PBAD activator-promoter. The yield of interleukin-10 delivered by the phage lysis system was too low for functional analysis whereas the fusion protein secreted by the hemolysin transporter proved to be biologically inactive. Moreover, partial processing of the fusion protein by the E. coli membrane protease OmpT had no effect on the protein’s functionality. Using the α-mating factor signal sequence, the yeast S. boulardii proved to be suitable for secretory expression of biologically active viral interleukin-10.