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1.  Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli 
Infection and Immunity  2014;82(11):4631-4642.
Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.
PMCID: PMC4249321  PMID: 25156739
2.  A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6 
PLoS ONE  2013;8(5):e64149.
Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.
PMCID: PMC3667844  PMID: 23737971
3.  Globosides but not isoglobosides can impact the development of invariant natural killer T cells and their interaction with dendritic cells1 
Recognition of endogenous lipid antigen(s) on CD1d is required for the development of invariant natural killer T (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control over-stimulation and deletion of iNKT cells in α-galactosidase A-deficient mice (αGalA−/−, human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this paper we generate a globotrihexosylceramide (Gb3) synthase deficient mouse (Gb3S−/−) and show that in αGalA−/−/Gb3S−/− double knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by high performance liquid chromatography. Furthermore we demonstrate that iGb3-deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the T cell receptor (TCR) complementarity determining region 3 of iNKT cells. Analyzing multiple gene targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective antigen presentation in αGalA−/− mice. Finally, we show that correction of globoside storage in αGalA−/− mice by crossing them with Gb3S−/− normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA−/−/Gb3S−/− mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover in αGalA−/− mice it is the Gb3-storage that is responsible for the decreased iNKT cell numbers and impeded antigen presentation on DCs.
PMCID: PMC3442250  PMID: 22875802
4.  Distribution and Phylogeny of Immunoglobulin-Binding Protein G in Shiga Toxin-Producing Escherichia coli and Its Association with Adherence Phenotypes▿  
Infection and Immunity  2010;78(8):3625-3636.
eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae-negative STEC strains but in none of 157 eae-positive STEC strains. The 36 eibG-positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H− and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG-positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes (eibG-α, eibG-β, and eibG-γ). Strains expressing EibG-α and EibG-β displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-γ adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG-α, eibG-β, and eibG-γ subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.
PMCID: PMC2916290  PMID: 20547747
5.  A new male sex-pheromone and novel cuticular cues for chemical communication in Drosophila 
Current biology : CB  2009;19(15):1245-1254.
In many insect species, cuticular hydrocarbons serve as pheromones that can mediate complex social behaviors. In Drosophila melanogaster, several hydrocarbons including the male sex pheromone 11-cis-vaccenyl acetate (cVA) and female-specific 7,11-dienes influence courtship behavior and can function as cues for short-term memory associated with the mating experience. Behavioral and physiological studies suggest that other unidentified chemical communication cues are likely to exist. To more fully characterize the hydrocarbon profile of the D. melanogaster cuticle, we applied direct ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry (UV-LDI-o-TOF MS) and analyzed the surface of intact fruit flies at a spatial resolution of approximately 200 μm.
We report the chemical and spatial characterization of 28 species of cuticular hydrocarbons, including a new major class of oxygen-containing compounds. Using UV-LDI MS, pheromones previously shown to be expressed exclusively by one sex, e.g. cVA, 7,11-heptacosadiene, and 7,11-nonacosadiene, appear to be found on both male and female flies. In males, cVA co-localizes at the tip of the ejaculatory bulb with a second acetylated hydrocarbon named CH503. We describe the chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and show one behavioral role for this compound as a long-lived inhibitor of male courtship. Like cVA, CH503 is transferred from males to females during mating. Unlike cVA, CH503 remains on the surface of females for at least 10 days.
Oxygenated hydrocarbons comprise one major previously undescribed class of compounds on the Drosophila cuticular surface. In addition to cVA, a newly-discovered long chain acetate, CH503, serves as a mediator of courtship-related chemical communication.
PMCID: PMC2726907  PMID: 19615904
6.  Shiga Toxin Receptor Gb3Cer/CD77: Tumor-Association and Promising Therapeutic Target in Pancreas and Colon Cancer 
PLoS ONE  2009;4(8):e6813.
Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.
Methodology/Principal Findings
Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.
Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.
PMCID: PMC2730034  PMID: 19714252
7.  Anaerobic Conditions Promote Expression of Sfp Fimbriae and Adherence of Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM to Human Intestinal Epithelial Cells▿  
The sfp gene cluster, unique to sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM strains, encodes fimbriae that mediate mannose-resistant hemagglutination in laboratory E. coli strains but are not expressed in wild-type SF EHEC O157:NM strains under standard laboratory conditions. We investigated whether Sfp fimbriae are expressed under conditions that mimic the intestinal environment and whether they contribute to the adherence of SF EHEC O157:NM strains to human intestinal epithelial cells. The transcription of sfpA (encoding the major fimbrial subunit) was upregulated in all strains investigated, and all expressed SfpA and possessed fimbriae that reacted with an anti-SfpA antibody when the strains were grown on solid media under anaerobic conditions. Sfp expression was absent under aerobic conditions and in liquid media. Sfp upregulation under anaerobic conditions was significantly higher on blood agar and a medium simulating the colonic environment than on a medium simulating the ileal environment (P < 0.05). The induction of Sfp fimbriae in SF E. coli O157:NM strains correlates with increased adherence to Caco-2 and HCT-8 cells. Our data indicate that the expression of Sfp fimbriae in SF E. coli O157:NM strains is induced under conditions resembling those of the natural site of infection and that Sfp fimbriae may contribute to the adherence of the organisms to human intestinal epithelium.
PMCID: PMC2258567  PMID: 18083855
8.  Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion 
Cytotechnology  2002;38(1-3):47-56.
On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment –capturing and rolling of leukocytes. A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor. In the smallest system 25.4 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 3.7 × 106 cells ml-1. Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 × 107 cells ml-1 in the 2 l bioreactor within 29 days. Large scale cultivation in a 100 l stirred tankbioreactor yielded 105.6 mg in three batches with a maximal viable cell density of 9.7 × 105 cells ml-1 within 15 days. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration. Purificationwas performed by affinity chromatography with rProtein A. Integrity of the E-selectin-IgG protein was checked with SDS PAGE. Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively. Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively. A concentation of 18.8 and 37.5 μg ml-1was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.
PMCID: PMC3449925  PMID: 19003086
adhesion; batch; CHO; endothelium; E-selectin; HL-60; HUVEC; inflammation; leukocytes; perfusion; serum free; spin filter
9.  Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα 
Environmental Microbiology  2011;13(5):1327-1341.
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 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 Leu235 and Ser236 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.
PMCID: PMC3472028  PMID: 21352460
10.  Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure 
The Journal of Pathology  2014;234(1):120-133.
The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22–44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31–17.60) mg/dl, lactate dehydrogenase 1944 (753–2792) U/l, platelets 33 (19–124)/nl and haemoglobin 6.2 (5.2–7.8) g/dl; median (range)], all patients were discharged after 33 (range 19–43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84–2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66–1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2-mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
PMCID: PMC4282478  PMID: 24909663
acute renal failure; acute tubular damage; electron microscopy; globoside (Gb3, CD77); Shigatoxin; Shigatoxin-producing Escherichia coli (STEC); thrombotic microangiopathy

Results 1-10 (10)