The Experts below are selected from a list of 7347 Experts worldwide ranked by ideXlab platform
Kirsten J Koymans - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3)
Veterinary Research, 2018Co-Authors: Kirsten J Koymans, Louris J Feitsma, Carla J C De Haas, Jos A G Van Strijp, Eric G Huizinga, Adinda Bisschop, Alex J. MccarthyAbstract:AbstractStaphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein–protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3–TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2–SSL3 interaction which adds to our understanding of staphylococcal host specificity.
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the tlr2 antagonist staphylococcal superantigen like protein 3 acts as a virulence factor to promote bacterial pathogenicity in vivo
Journal of Innate Immunity, 2017Co-Authors: Kirsten J Koymans, Oliver Goldmann, Christofer Karlsson, Wiedjai Sital, Robert Thanert, Adinda Bisschop, Manouk VrielingAbstract:Toll-like receptor (TLR) signaling is important in the initiation of immune responses and subsequent instigation of adaptive immunity. TLR2 recognizes bacterial lipoproteins and plays a central role in the host defense against bacterial infections, including those caused by Staphylococcus aureus. Many studies have demonstrated the importance of TLR2 in murine S. aureus infection. S. aureus evades TLR2 activation by secreting two proteins, staphylococcal superantigen-like protein 3 (SSL3) and 4 (SSL4). In this study, we demonstrate that antibodies against SSL3 and SSL4 are found in healthy individuals, indicating that humans are exposed to these proteins during S. aureus colonization or infection. To investigate the TLR2-antagonistic properties of SSL3 and SSL4, we compared the infection with wild-type and SSL3/4 knockout S. aureus strains in an intravenous murine infection model. Direct evaluation of the contribution of SSL3/4 to infection pathogenesis was hindered by the fact that the SSLs were not expressed in the murine system. To circumvent this limitation, an SSL3-overproducing strain (pLukM-SSL3) was generated, resulting in constitutive expression of SSL3. pLukM-SSL3 exhibited increased virulence compared to the parental strain in a murine model that was found to be TLR2 dependent. Altogether, these data indicate that SSL3 contributes to S. aureus virulence in vivo.
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structural basis for inhibition of tlr2 by staphylococcal superantigen like protein 3 ssl3
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Kirsten J Koymans, Louris J Feitsma, Harma T C Brondijk, Piet C Aerts, Eddie Lukkien, Philip Lossl, Kok P M Van Kessel, Carla J C De Haas, Jos A G Van Strijp, Eric G HuizingaAbstract:Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.
Andrew Szilagyi - One of the best experts on this subject based on the ideXlab platform.
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manipulation of intestinal microbial flora for therapeutic benefit in inflammatory bowel diseases review of clinical trials of probiotics pre biotics and synbiotics
Reviews on Recent Clinical Trials, 2008Co-Authors: Debra Heilpern, Andrew SzilagyiAbstract:: Pathogenesis of Inflammatory Bowel Diseases(Ulcerative Colitis, Crohn's disease and Pouchitis) includes an abnormal immunological response to disturbed intestinal microflora. Therapeutic strategies are designed to intervene in these abnormal host microbial communications. A novel approach in the last decade has been to use other bacteria or selective foods to induce beneficial bacteria to normalize inflammation. In this review we discuss rationale for such use and describe 46 clinical trials gleaned from the literature. Reports are divided into type, indications, and agents used. The search revealed 15 nonrandomized and 31 randomized trials. Of the latter 23 were double-blind and 8 were open-label randomized controlled. In 32 of the total, different probiotics were used, while 10 and 4 used different prebiotics or synbiotics respectively. In 14 nonrandomized trials, outcome was successful. In the randomized controlled trials 12 of 16 ulcerative colitis but only 2 of Crohn's disease trials of biotic therapy were successful. No superiority of any probiotic was clearly evident, but a multi-agent mixture, VSL3# may be better suited in ulcerative colitis and pouchitis while the probiotic Lactobacillus rhamnosus GG appears less useful in inflammatory bowel disease, especially Crohn's disease. Further studies with uniform stringent criteria are needed to provide proof of this therapy in inflammatory bowel disease.
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failure to improve parameters of lactose maldigestion using the multiprobiotic product VSL3 in lactose maldigesters a pilot study
Canadian Journal of Gastroenterology & Hepatology, 2004Co-Authors: Rose Yesovitch, Albert Cohen, Andrew SzilagyiAbstract:Lactose maldigestion is a common genetic trait in up to 70% of the world's population. In these subjects, the ingestion of lactose may lead to prebiotic effects which can be confirmed by measurement of breath hydrogen. After a period of continuous lactose ingestion, colonic bacterial adaptation is measurable as improved parameters of lactose digestion. There may be inherent benefits in this process of adaptation which may protect against some diseases. We attempt to link therapeutically beneficial probiotics (VSL3, Seaford Pharmaceuticals Inc, Ontario) with improvement in parameters of lactose maldigestion. Two groups of five subjects with maldigestion were fed one or four packets of VSL3 (one packet containing 450 x 10(9) live bacteria) before testing and then 17 days later. A 50 g lactose challenge was carried out before and after feeding. While there was a trend toward increasing rather than reducing of summed breath hydrogen, no statistically significant changes were observed between results from before testing and those from testing 17 days later. The authors conclude that direct consumption of the probiotic VSL3 may not improve parameters of lactose maldigestion without metabolic activation. In its present format, therefore, the test for colonic adaptation cannot be used to demonstrate direct bacterial embedding with VSL3.
Simone Fulda - One of the best experts on this subject based on the ideXlab platform.
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oncogenic ras mutants confer resistance of rms13 rhabdomyosarcoma cells to oxidative stress induced ferroptotic cell death
Frontiers in Oncology, 2015Co-Authors: Christina Schott, Ulrike Graab, Nicole Cuvelier, Heidi Hahn, Simone FuldaAbstract:Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS13 cells. Here, we report that ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 cells from oxidative stress-induced cell death. RMS13 cells engineered to express NRAS12V, KRAS12V, or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system [Formula: see text] that blocks GSH synthesis. Notably, addition of Ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programed cell death. Furthermore, RMS13 cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS13 cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS13 cells has important implications for the development of targeted therapies for RMS.
Eric G Huizinga - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3)
Veterinary Research, 2018Co-Authors: Kirsten J Koymans, Louris J Feitsma, Carla J C De Haas, Jos A G Van Strijp, Eric G Huizinga, Adinda Bisschop, Alex J. MccarthyAbstract:AbstractStaphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein–protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3–TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2–SSL3 interaction which adds to our understanding of staphylococcal host specificity.
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structural basis for inhibition of tlr2 by staphylococcal superantigen like protein 3 ssl3
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Kirsten J Koymans, Louris J Feitsma, Harma T C Brondijk, Piet C Aerts, Eddie Lukkien, Philip Lossl, Kok P M Van Kessel, Carla J C De Haas, Jos A G Van Strijp, Eric G HuizingaAbstract:Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.
Karl Kunzelmann - One of the best experts on this subject based on the ideXlab platform.
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tmem16f anoctamin 6 in ferroptotic cell death
Cancers, 2019Co-Authors: Jiraporn Ousingsawat, Rainer Schreiber, Karl KunzelmannAbstract:Ca2+ activated Cl− channels (TMEM16A; ANO1) support cell proliferation and cancer growth. Expression of TMEM16A is strongly enhanced in different types of malignomas. In contrast, TMEM16F (ANO6) operates as a Ca2+ activated chloride/nonselective ion channel and scrambles membrane phospholipids to expose phosphatidylserine at the cell surface. Both phospholipid scrambling and cell swelling induced through activation of nonselective ion currents appear to destabilize the plasma membrane thereby causing cell death. There is growing evidence that activation of TMEM16F contributes to various forms of regulated cell death. In the present study, we demonstrate that ferroptotic cell death, occurring during peroxidation of plasma membrane phospholipids activates TMEM16F. Ferroptosis was induced by erastin, an inhibitor of the cystine-glutamate antiporter and RSL3, an inhibitor of glutathione peroxidase 4 (GPX4). Cell death was largely reduced in the intestinal epithelium, and in peritoneal macrophages isolated from mice with tissue-specific knockout of TMEM16F. We show that TMEM16F is activated during erastin and RSL3-induced ferroptosis. In contrast, inhibition of ferroptosis by ferrostatin-1 and by inhibitors of TMEM16F block TMEM16F currents and inhibit cell death. We conclude that activation of TMEM16F is a crucial component during ferroptotic cell death, a finding that may be useful to induce cell death in cancer cells.
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TMEM16F/Anoctamin 6 in Ferroptotic Cell Death
MDPI AG, 2019Co-Authors: Jiraporn Ousingsawat, Rainer Schreiber, Karl KunzelmannAbstract:Ca2+ activated Cl− channels (TMEM16A; ANO1) support cell proliferation and cancer growth. Expression of TMEM16A is strongly enhanced in different types of malignomas. In contrast, TMEM16F (ANO6) operates as a Ca2+ activated chloride/nonselective ion channel and scrambles membrane phospholipids to expose phosphatidylserine at the cell surface. Both phospholipid scrambling and cell swelling induced through activation of nonselective ion currents appear to destabilize the plasma membrane thereby causing cell death. There is growing evidence that activation of TMEM16F contributes to various forms of regulated cell death. In the present study, we demonstrate that ferroptotic cell death, occurring during peroxidation of plasma membrane phospholipids activates TMEM16F. Ferroptosis was induced by erastin, an inhibitor of the cystine-glutamate antiporter and RSL3, an inhibitor of glutathione peroxidase 4 (GPX4). Cell death was largely reduced in the intestinal epithelium, and in peritoneal macrophages isolated from mice with tissue-specific knockout of TMEM16F. We show that TMEM16F is activated during erastin and RSL3-induced ferroptosis. In contrast, inhibition of ferroptosis by ferrostatin-1 and by inhibitors of TMEM16F block TMEM16F currents and inhibit cell death. We conclude that activation of TMEM16F is a crucial component during ferroptotic cell death, a finding that may be useful to induce cell death in cancer cells
