23 results on '"Grijpstra J"'
Search Results
2. Capsule biogenesis in Cryptococcus neoformans
- Author
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Grijpstra, J., Molecular Microbiology, Dep Biologie, Wösten, Han, and de Cock, Hans
- Subjects
Cell biology ,Biologie/Milieukunde (BIOL) ,Molecular biology ,International (English) ,Life sciences ,Microbiology - Abstract
Cryptococcus neoformans is a human pathogen that mainly causes disease in immuno-compromised individuals. The yeast cells or basidiospores spread through dissemination from environmental sources, for example pigeon droppings and dust, and causes a systemic infection, cryptococcosis. A severe clinical manifestation of cryptococcosis is meningo-encephalitis, but also infection of other organs like the lungs, and skin have been reported. Since the increased incidence of AIDS due to the spread of the HIV virus, especially in third world countries, C. neoformans has become one of the most emerging opportunistic pathogens in hospitals. In fact, it is one of the leading causes of death in patients suffering from AIDS. The incidence of cryptococcosis is also increasing in the Western world especially due to use of immunosuppressive drugs and increasing numbers of organ transplantations. The surface of the C. neoformans yeast cell can be surrounded by a thick capsule. This capsule is mainly composed of the polysaccharides GXM and GalXM. GalXM consists of the monosaccharides mannose, xylose and glucuronic acid, whereas GXM contains galactose, mannose and xylose. The capsule is critical for pathogenesis and harbors components that have immuno-modulatory properties. This and the physical properties of the capsule makes it a barrier for the immune system to reach receptors on the cell wall. Mutants have been isolated that have an acapsular phenotype. These mutants of the serotype A strain of C. neoformans are called CAP10, CAP59, CAP60 and CAP64. CAP67 is a serotype D mutant strain. The genes that are affected in these strains have been identified, but the exact role of the CAP genes is not clear. In this Thesis biosynthesis of the capsule and its role in pathogenesis was studied. This was done by investigating the content and the structure of the capsule, by looking at the genes involved in capsule biogenesis and by investigating the effect of mutants affected in capsule biogenesis on human dendritic cells. This is not only interesting from a fundamental point of view, it may also identify targets that can be used to improve treatment of cryptococcosis in the future.
- Published
- 2008
3. ArcGIS tools for the prediction and evaluation of terrestrial ecosystems
- Author
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Raterman, B., Grijpstra, J., and Witte, J.P.M.
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WIMEK ,Soil Physics, Ecohydrology and Groundwater Management ,Life Science ,Leerstoelgroep Bodemnatuurkunde, ecohydrologie en grondwaterbeheer - Published
- 2004
4. Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups
- Author
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Harmsen, HJM, Wildeboer-Veloo, ACM, Grijpstra, J, Knol, J, Degener, JE, Welling, GW, Groningen Institute for Gastro Intestinal Genetics and Immunology (3GI), and Man, Biomaterials and Microbes (MBM)
- Subjects
TARGETED OLIGONUCLEOTIDE PROBES ,HUMAN FECAL SAMPLES ,PHENOTYPIC EVIDENCE ,EUBACTERIUM-LENTUM ,SEQUENCE-ANALYSIS ,COMB. NOV ,GEN. NOV ,IN-SITU HYBRIDIZATION ,RIBOSOMAL-RNA ,COMMUNITIES - Abstract
Two 16S rRNA-targeted probes were developed: one for the Coriobacterium group and the other for the Atopobium cluster (which comprises most of the Coriobacteriaceae species, including the Coriobacterium group). The new probes were based on sequences of three new Coriobacteriaceae strains isolated from human feces and clinical material and sequences from databases. Application of the probes to fecal samples showed that formula-fed infants had higher numbers of Coriobacterium group cells in their feces than breast-fed infants. In addition, based on the presented results, it is hypothesized that with the increasing age of a person, the diversity of Atopobium cluster species present in the feces increases.
- Published
- 2000
5. Nut en noodzaak van OBN voor de Nederlandse natuur na 2010
- Author
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Grijpstra, J., Wamelink, G.W.W., Meuleman, A.F.M., and van Dobben, H.F.
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WIMEK ,Life Science ,Plantenecologie en Natuurbeheer ,Plant Ecology and Nature Conservation ,Wageningen Environmental Research - Published
- 2000
6. Perspectieven voor gezinsbedrijven in het zuidelijk zandgebied
- Author
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Grijpstra, J., Preuter, H., and Peters Rit, C.J.
- Subjects
bodemtextuur ,Research and Advisory Institute for Field Crop and Grassland Husbandry ,netherlands ,soil texture ,farms ,nederland ,small farms ,landbouwbedrijven ,bedrijfssystemen ,bodemtypen ,farming systems ,soil types ,Proefstation voor Akker- en Weidebouw ,kleine landbouwbedrijven - Published
- 1967
7. De rentabiliteit van hooiventilatie
- Author
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Grijpstra, J., Wind, T.T., and de Widt, R.A.
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Wageningen Economic Research ,drying ,drogen - Published
- 1960
8. De landbouw in het zuidelijk zandgebied
- Author
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Grijpstra, J., Preuter, H., and Peters Rit, C.J.
- Subjects
noord-brabant ,limburg ,Research and Advisory Institute for Field Crop and Grassland Husbandry ,Proefstation voor Akker- en Weidebouw ,netherlands ,agrarische bedrijfsvoering ,farm management ,nederland - Published
- 1966
9. Inventarisatie arbeidsonderzoek
- Author
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Grijpstra, J.
- Subjects
Wageningen Economic Research ,Life Science - Published
- 1960
10. Begrotingen voor veenkoloniale akkerbouwbedrijven in de Krim (Ov.)
- Author
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Aaftink, E. and Grijpstra, J.
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farm accounting ,schattingen ,accounting ,netherlands ,arable farming ,nederland ,veldgewassen ,financial planning ,boekhouding van landbouwbedrijf ,farming systems ,boekhouden ,boekhouding ,Research and Advisory Institute for Field Crop and Grassland Husbandry ,estimates ,financiële planning ,farm accounts ,farms ,overijssel ,landbouwbedrijven ,field crops ,bedrijfssystemen ,Proefstation voor Akker- en Weidebouw ,akkerbouw ,rekeningen van landbouwbedrijf ,book-keeping - Published
- 1964
11. Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry.
- Author
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Di Marco F, Hipgrave Ederveen AL, van Schaick G, Moran AB, Domínguez-Vega E, Nicolardi S, Blöchl C, Koeleman CA, Danuser R, Al Kaabi A, Dotz V, Grijpstra J, Beurret M, Anish C, and Wuhrer M
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- Glycosylation, Mass Spectrometry methods, Vaccines, Conjugate chemistry, Vaccines, Conjugate immunology, Chromatography, Liquid methods, Chromatography, Reverse-Phase methods, Glycoconjugates chemistry, Glycoconjugates immunology, Bacterial Vaccines immunology, Bacterial Vaccines chemistry, Glycopeptides chemistry, Glycopeptides analysis
- Abstract
Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.B.M., R.D., V.D., A.A.K., J.G., M.B., and C.A. are employees of Janssen Vaccines. All other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2024
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12. Deletion of the CAP10 gene of Cryptococcus neoformans results in a pleiotropic phenotype with changes in expression of virulence factors.
- Author
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Tefsen B, Grijpstra J, Ordonez S, Lammers M, van Die I, and de Cock H
- Subjects
- Cryptococcus neoformans cytology, Exosomes ultrastructure, Fungal Proteins metabolism, Gene Expression Profiling, Phenotype, Cryptococcus neoformans genetics, Fungal Proteins genetics, Gene Deletion, Gene Expression, Gene Expression Regulation, Fungal, Genetic Pleiotropy, Virulence Factors biosynthesis
- Abstract
The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is an important virulence factor for this yeast-like fungus. Previously, we had shown that disruption of the CAP10 gene, encoding a putative xylosyltransferase, results in mutant cells that lack most of the capsular polysaccharides on the cell surface, but do not show a typical acapsular phenotype. In contrast to the acapsular cap59 mutant, cap10 did not induce maturation of dendritic cells when exposed to components of the immune system. In order to gain further insight into the causes of this phenotype displayed by the cap10 mutant, we performed a more in-depth phenotypic analysis of the cell wall and surface structures of this mutant compared to the wild type strain and acapsular mutant cap59. Moreover, we analyzed the cap10 mutant and the wild type strain for differential gene expression of, amongst others, enzymes that are involved in biogenesis of cell wall and capsule components. We conclude that a mutation in the CAP10 gene results in a pleiotropic phenotype with effects on different cellular processes affecting, amongst others, cell size, expression of virulence factors and size of extracellular vesicles., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)
- Published
- 2014
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13. Involvement of Neisseria meningitidis lipoprotein GNA2091 in the assembly of a subset of outer membrane proteins.
- Author
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Bos MP, Grijpstra J, Tommassen-van Boxtel R, and Tommassen J
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- Amino Acid Sequence, Bacterial Outer Membrane Proteins chemistry, Conserved Sequence, Gene Deletion, Lipoproteins genetics, Lipoproteins metabolism, Molecular Sequence Data, Mutagenesis, Neisseria meningitidis, Serogroup B genetics, Phenotype, Porins metabolism, Protein Structure, Tertiary, Bacterial Outer Membrane Proteins metabolism, Bacterial Vaccines metabolism, Neisseria meningitidis, Serogroup B metabolism
- Abstract
GNA2091 of Neisseria meningitidis is a lipoprotein of unknown function that is included in the novel 4CMenB vaccine. Here, we investigated the biological function and the subcellular localization of the protein. We demonstrate that GNA2091 functions in the assembly of outer membrane proteins (OMPs) because its absence resulted in the accumulation of misassembled OMPs. Cell fractionation and protease accessibility experiments showed that the protein is localized at the periplasmic side of the outer membrane. Pulldown experiments revealed that it is not stably associated with the β-barrel assembly machinery, the previously identified complex for OMP assembly. Thus, GNA2091 constitutes a novel outer membrane-based lipoprotein required for OMP assembly. Furthermore, its location at the inner side of the outer membrane indicates that protective immunity elicited by this antigen cannot be due to bactericidal or opsonic activity of antibodies., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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14. Species-specificity of the BamA component of the bacterial outer membrane protein-assembly machinery.
- Author
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Volokhina EB, Grijpstra J, Beckers F, Lindh E, Robert V, Tommassen J, and Bos MP
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- Bacterial Outer Membrane Proteins metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli Proteins metabolism, Genetic Complementation Test, Gram-Negative Bacteria metabolism, Immunoblotting, Plasmids genetics, Protein Conformation, Reverse Transcriptase Polymerase Chain Reaction, Species Specificity, Bacterial Outer Membrane Proteins genetics, Escherichia coli Proteins genetics, Gram-Negative Bacteria genetics, Multiprotein Complexes genetics, Multiprotein Complexes metabolism
- Abstract
The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.
- Published
- 2013
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15. Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.
- Author
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Stork M, Grijpstra J, Bos MP, Mañas Torres C, Devos N, Poolman JT, Chazin WJ, and Tommassen J
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- Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Humans, Ion Transport genetics, Ion Transport immunology, Iron immunology, Iron metabolism, Leukocyte L1 Antigen Complex immunology, Leukocyte L1 Antigen Complex metabolism, Manganese immunology, Manganese metabolism, Neisseria meningitidis genetics, Neisseria meningitidis metabolism, Neutrophils immunology, Neutrophils metabolism, Zinc metabolism, Bacterial Outer Membrane Proteins immunology, Bacterial Proteins immunology, Immune Evasion, Neisseria meningitidis immunology, Zinc immunology
- Abstract
The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.
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- 2013
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16. Autotransporter secretion: varying on a theme.
- Author
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Grijpstra J, Arenas J, Rutten L, and Tommassen J
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Cell Membrane chemistry, Cell Membrane genetics, Cell Membrane metabolism, Gram-Negative Bacteria chemistry, Gram-Negative Bacteria genetics, Membrane Transport Proteins chemistry, Membrane Transport Proteins genetics, Protein Structure, Tertiary, Protein Transport, Bacterial Proteins metabolism, Bacterial Secretion Systems, Gram-Negative Bacteria metabolism, Membrane Transport Proteins metabolism
- Abstract
Autotransporters are widely distributed among Gram-negative bacteria. They can have a large variety of functions and many of them have a role in virulence. They are synthesized as large precursors with an N-terminal signal sequence that mediates transport across the inner membrane via the Sec machinery and a translocator domain that mediates the transport of the connected passenger domain across the outer membrane to the bacterial cell surface. Like integral outer membrane proteins, the translocator domain folds in a β-barrel structure and requires the Bam machinery for its insertion into the outer membrane. After transport across the outer membrane, the passenger may stay connected via the translocator domain to the bacterial cell surface or it is proteolytically released into the extracellular milieu. Based on the size of the translocator domain and its position relative to the passenger in the precursor, autotransporters are divided into four sub-categories. We review here the current knowledge of the biogenesis, structure and function of various autotransporters., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)
- Published
- 2013
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17. Lipidation of the autotransporter NalP of Neisseria meningitidis is required for its function in the release of cell-surface-exposed proteins.
- Author
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Roussel-Jazédé V, Grijpstra J, van Dam V, Tommassen J, and van Ulsen P
- Subjects
- Alanine genetics, Alanine metabolism, Amino Acid Substitution, Cysteine genetics, Cysteine metabolism, Kinetics, Lipid Metabolism, Membrane Transport Proteins genetics, Mutagenesis, Site-Directed, Mutant Proteins genetics, Mutant Proteins metabolism, Serine Endopeptidases genetics, Membrane Transport Proteins metabolism, Neisseria meningitidis metabolism, Protein Processing, Post-Translational, Serine Endopeptidases metabolism
- Abstract
Autotransporters of Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain and the secreted passenger domain in between. The autotransporter NalP of Neisseria meningitidis includes a protease domain that facilitates the release of several immunogenic proteins from the cell surface into the extracellular milieu. Rather exceptionally among autotransporters, NalP is a lipoprotein. We investigated the role of lipidation in the biogenesis and function of the protein. To this end, the N-terminal cysteine, which is lipidated in the wild-type protein, was substituted by alanine. Like the wild-type protein, the mutant protein was secreted into the medium, demonstrating that lipidation is not required for biogenesis of the protein. However, the non-lipidated NalP variant had a drastically reduced capacity to cleave its substrate proteins from the cell surface, suggesting that the lipid moiety is important for function. Kinetic experiments demonstrated that the autocatalytic processing of the non-lipidated protein at the cell surface was much faster than that of the wild-type protein. Thus, the lipid moiety delays the release of NalP from the cell surface, thereby allowing it to release other surface-exposed proteins into the milieu.
- Published
- 2013
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18. Assembly of bacterial outer membrane proteins.
- Author
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Grijpstra J, Bos MP, and Tommassen J
- Subjects
- Blotting, Western, Electrophoresis, Polyacrylamide Gel, Electrophoretic Mobility Shift Assay, Bacterial Outer Membrane Proteins metabolism
- Abstract
Various methods that are routinely used to study the subcellular localization of membrane proteins in wild-type Gram-negative bacteria fall short in genetic studies addressing the biogenesis of outer membrane proteins (OMPs). Here, we describe three biochemical methods that can be used in such studies to evaluate the proper assembly of OMPs into the outer membrane. The methods are based on (1) the differential electrophoretic mobility of folded and nonnative OMPs, (2) the intrinsically high protease resistance of folded OMPs, and (3) the observation that integral membrane proteins are not extracted from the membrane in solutions containing high concentrations of urea.
- Published
- 2013
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19. Role of the periplasmic chaperones Skp, SurA, and DegQ in outer membrane protein biogenesis in Neisseria meningitidis.
- Author
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Volokhina EB, Grijpstra J, Stork M, Schilders I, Tommassen J, and Bos MP
- Subjects
- Bacterial Outer Membrane Proteins genetics, Escherichia coli metabolism, Molecular Chaperones genetics, Mutation, Neisseria meningitidis genetics, Porins, Bacterial Outer Membrane Proteins metabolism, Gene Expression Regulation, Bacterial physiology, Molecular Chaperones metabolism, Neisseria meningitidis metabolism
- Abstract
The periplasmic chaperones Skp, SurA, and DegP are implicated in the biogenesis of outer membrane proteins (OMPs) in Escherichia coli. Here, we investigated whether these chaperones exert similar functions in Neisseria meningitidis. Although N. meningitidis does not contain a homolog of the protease/chaperone DegP, it does possess a homolog of another E. coli protein, DegQ, which can functionally replace DegP when overproduced. Hence, we examined whether in N. meningitidis, DegQ acts as a functional homolog of DegP. Single skp, surA, and degQ mutants were easily obtained, showing that none of these chaperones is essential in N. meningitidis. Furthermore, all combinations of double mutants were generated and no synthetic lethality was observed. The absence of SurA or DegQ did not affect OMP biogenesis. In contrast, the absence of Skp resulted in severely lower levels of the porins PorA and PorB but not of other OMPs. These decreased levels were not due to proteolytic activity of DegQ, since porin levels remained low in a skp degQ double mutant, indicating that neisserial DegQ is not a functional homolog of E. coli DegP. The absence of Skp resulted in lower expression of the porB gene, as shown by using a P(porB)-lacZ fusion. We found no cross-species complementation when Skp of E. coli or N. meningitidis was heterologously expressed in skp mutants, indicating that Skp functions in a species-specific manner. Our results demonstrate an important role for Skp but not for SurA or DegQ in OMP biogenesis in N. meningitidis.
- Published
- 2011
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20. The Cryptococcus neoformans cap10 and cap59 mutant strains, affected in glucuronoxylomannan synthesis, differentially activate human dendritic cells.
- Author
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Grijpstra J, Tefsen B, van Die I, and de Cock H
- Subjects
- B7-1 Antigen metabolism, B7-2 Antigen metabolism, Cell Differentiation immunology, Cells, Cultured, Cryptococcus neoformans genetics, Cryptococcus neoformans pathogenicity, Culture Media, Conditioned, Cytokines metabolism, Flow Cytometry, Fungal Proteins genetics, Fungal Proteins metabolism, Humans, Immunohistochemistry, Polysaccharides biosynthesis, Cryptococcus neoformans immunology, Dendritic Cells immunology, Fungal Proteins immunology, Polysaccharides immunology
- Abstract
The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides: glucuronoxylomannan and galactoxylomannan. The presence of this polysaccharide capsule was previously shown to interfere with maturation of human dendritic cells (DCs), possibly by shielding cell-wall components from interacting with these host immune cells. Here we show that two mutant strains of C. neoformans, both lacking a visible capsule due to a defect in glucuronoxylomannan synthesis, differentially activate human monocyte-derived DCs. Cells from a cap59 mutant, but not of a cap10 mutant strain, induce maturation of DCs as indicated by an increase in the expression of the costimulatory molecules CD80 and CD86, and production of the cytokines interleukin (IL)-10, IL-12p40 and tumor necrosis factor alpha. Interestingly, cap59 mutant cells reassociated with a concentrated culture medium of wild-type C. neoformans had lost their capacity to induce DC maturation. Summarizing, our data suggest that glucuronoxylomannan confers properties to the capsule that protect the fungus against activation of DCs; however, the presence of intact glucuronoxylomannan is not an absolute requirement to prevent activation of DCs.
- Published
- 2009
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21. Production of extracellular polysaccharides by CAP mutants of Cryptococcus neoformans.
- Author
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Grijpstra J, Gerwig GJ, Wösten H, Kamerling JP, and de Cock H
- Subjects
- Biological Transport, Cryptococcus neoformans chemistry, Extracellular Space chemistry, Fungal Proteins metabolism, Polysaccharides chemistry, Polysaccharides isolation & purification, Cryptococcus neoformans genetics, Cryptococcus neoformans metabolism, Extracellular Space metabolism, Fungal Proteins genetics, Mutation, Polysaccharides metabolism
- Abstract
The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional (1)H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.
- Published
- 2009
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22. Septal pore cap protein SPC18, isolated from the basidiomycetous fungus Rhizoctonia solani, also resides in pore plugs.
- Author
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van Driel KG, van Peer AF, Grijpstra J, Wösten HA, Verkleij AJ, Müller WH, and Boekhout T
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Fungal Proteins chemistry, Fungal Proteins genetics, Glycoproteins chemistry, Glycoproteins genetics, Hyphae chemistry, Hyphae genetics, Hyphae ultrastructure, Molecular Sequence Data, Protein Transport, Rhizoctonia chemistry, Rhizoctonia genetics, Rhizoctonia ultrastructure, Fungal Proteins metabolism, Glycoproteins metabolism, Hyphae metabolism, Rhizoctonia metabolism
- Abstract
The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.
- Published
- 2008
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23. Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups.
- Author
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Harmsen HJ, Wildeboer-Veloo AC, Grijpstra J, Knol J, Degener JE, and Welling GW
- Subjects
- Actinobacteria genetics, Actinobacteria isolation & purification, Adult, Aged, Aged, 80 and over, Breast Feeding, Child, Child, Preschool, Humans, In Situ Hybridization, Fluorescence, Infant, Infant Food, Infant, Newborn, Middle Aged, Milk, Human, Molecular Sequence Data, Phenotype, Actinobacteria classification, Aging, Feces microbiology, Phylogeny, RNA, Ribosomal, 16S genetics
- Abstract
Two 16S rRNA-targeted probes were developed: one for the Coriobacterium group and the other for the Atopobium cluster (which comprises most of the Coriobacteriaceae species, including the Coriobacterium group). The new probes were based on sequences of three new Coriobacteriaceae strains isolated from human feces and clinical material and sequences from databases. Application of the probes to fecal samples showed that formula-fed infants had higher numbers of Coriobacterium group cells in their feces than breast-fed infants. In addition, based on the presented results, it is hypothesized that with the increasing age of a person, the diversity of Atopobium cluster species present in the feces increases.
- Published
- 2000
- Full Text
- View/download PDF
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