Jan Tommassen

Universiteit Utrecht, Utrecht, Utrecht, Netherlands

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Publications (111)534.44 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Forty-nine clinical Escherichia coli isolates, both extended-spectrum β-lactamase (ESBL) negative and ESBL positive, were studied to investigate whether increased AmpC expression is a mechanism involved in cefoxitin resistance and if this influences the third-generation cephalosporin activity. Nine of 33 (27.2%) cefoxitin-resistant (minimum inhibitory concentration [MIC] >8 mg/L) isolates showed hyperproduction of chromosomal AmpC (c-AmpC) based on (1) at least two positive tests using AmpC inhibitors, (2) mutations in the promoter/attenuator regions, and (3) a 6.1- to 163-fold increase in c-ampC expression by quantitative reverse transcription-polymerase chain reaction. In ESBL-negative isolates, MICs of ceftazidime and cefotaxime were mostly above the wild-type (WT) level, but below the S/I breakpoint (EUCAST guideline), except for one isolate with MICs of 4 mg/L. No plasmid-mediated AmpCs were found. Periplasmic extracts of nine c-AmpC hyperproducers were preincubated with or without cefuroxime or ceftazidime and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cefuroxime and ceftazidime were stable to hydrolysis but acted as inhibitors of the enzyme. None of these isolates showed loss of porins. Thus, cefoxitin resistance has low specificity for detecting upregulated c-AmpC production. c-AmpC hyperproducing E. coli is mostly still susceptible to third-generation cephalosporins but less than WT E. coli. Surveillance of cefoxitin-resistant E. coli to monitor developments in the activity of third-generation cephalosporins against c-AmpC hyperproducers is warranted.
    Microbial drug resistance (Larchmont, N.Y.) 09/2014; · 1.99 Impact Factor
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    ABSTRACT: Like all classical monomeric autotransporters, IgA protease of Neisseria meningitidis is a modular protein consisting of an N-terminal signal sequence, a passenger domain, and a C-terminal translocator domain (TD) that assists in the secretion of the passenger domain across the outer membrane. The passenger of IgA protease consists of three separate domains, the protease domain, the γ-peptide, and the α-peptide that contains nuclear localization signals (NLS). The protease domain is released into the extracellular milieu either via autocatalytic processing or via cleavage by another autotransporter, NalP, expression of which is phase variable. NalP-mediated cleavage results in the release of a passenger that includes the α- and γ-peptides. Here, we studied the fate of the α-peptide when NalP is not expressed and observed strain-dependent differences. In meningococcal strains where the α-peptide contains a single NLS, the α-peptide remained covalently attached to the TD and was detected at the cell surface. In other strains, the α-peptide contains four NLS and is separated from the TD by an IgA protease autoproteolytic cleavage site. In many of those cases, the α-peptide was found non-covalently associated with the cells as a separate polypeptide. The cell surface association of the α-peptides may be physiologically relevant. We report a novel function for the α-peptide, i.e. the binding of heparin, an immune-modulatory molecule that in the host is found in the extracellular matrix and connected to cell surfaces.
    Microbiology (Reading, England). 08/2014;
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    ABSTRACT: Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilmsadhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosainfections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell-cell and cell-surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.
    Journal of Biotechnology 07/2014; · 3.18 Impact Factor
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    ABSTRACT: Autotransporters are proteins secreted by Gram-negative bacteria that often play a role in virulence. Eight different autotransporters have been identified in Neisseria meningitidis but only six of them have been characterized. AutA is one of the remaining autotransporters. Its expression remains controversial. Here, we show that the autA gene is present in many neisserial species but its expression is often disrupted by various genetic features; however, it is expressed in certain strains of N. meningitidis. By sequencing the autA gene in large panels of disease isolates and Western blot analysis, we demonstrated that AutA expression is prone to phase variation at AAGC nucleotide repeats located within the DNA encoding the signal sequence. AutA is not secreted into the extracellular medium but remains associated with the bacterial cell surface. We further demonstrate that AutA expression induces autoaggregation in a process that, dependent on the particular strain, may require extracellular DNA (eDNA). This property influences the organization of bacterial communities like lattices and biofilms. In vitro assays evidenced that AutA is a self-associating autotransporter that binds DNA. We suggest that AutA-mediated autoaggregation might be particularly important for colonization and persistence of the pathogen in the nasopharynx of the host.
    Environmental Microbiology 07/2014; · 6.24 Impact Factor
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    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) since 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. Pull-down experiments revealed that it is not stably associated with the BAM complex, the previously identified machinery 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.
    Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
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    ABSTRACT: The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical-shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.
    Journal of Molecular Biology 02/2014; · 3.91 Impact Factor
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    ABSTRACT: Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis was previously described that showed increased sensitivity to hydrophobic toxic compounds suggesting a breach in this permeability barrier. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants which produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstream located pilE gene resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered.
    Journal of bacteriology 12/2013; · 3.94 Impact Factor
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    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 Zn(2+) and Mn(2+) 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.
    PLoS Pathogens 10/2013; 9(10):e1003733. · 8.14 Impact Factor
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    ABSTRACT: Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis, several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA-related gene cassettes, called tpsC, are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be. We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination. Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.
    BMC Genomics 09/2013; 14(1):622. · 4.40 Impact Factor
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    ABSTRACT: A liver-transplant patient was admitted with cholangitis for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli, but, during admission a carbapenem-resistant (CR) E. coli was isolated. Analysis of the outer-membrane-protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed blaCTX-M-15 and blaOXA-1. The CR E. coli additionally harbored a blaCMY-2 and demonstrated a >15-fold increased β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance.In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer-membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the high amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 β-lactamase plays a role in carbapenem resistance.
    Antimicrobial Agents and Chemotherapy 06/2013; · 4.57 Impact Factor
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    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.
    Research in Microbiology 04/2013; · 2.89 Impact Factor
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    ABSTRACT: Neisseria meningitidis serogroup B (MenB) is a major cause of bacterial sepsis and meningitis, with the highest disease burden in young children. Available vaccines are based on outer membrane vesicles (OMVs) obtained from wild-type strains. However, particularly in toddlers and infants, they confer protection mostly against strains expressing the homologous PorA, a major and variable outer membrane protein. In the quest for alternative vaccine antigens able to provide broad MenB strain coverage in the younger populations, but potentially also across all age groups, ZnuD, a protein expressed under zinc-limiting conditions, may be considered as a promising candidate. Here, we have investigated the potential value of ZnuD and show that it is a conserved antigen expressed by all MenB strains tested except for some strains of clonal complex ST-8. In mice and guinea-pigs immunized with ZnuD-expressing OMVs, antibodies were elicited that were able to trigger complement-mediated killing of all the MenB strains and serogroup A, C, and Y strains tested, when grown under zinc limitation. ZnuD is also expressed during infection, since anti-ZnuD antibodies were detected in sera from patients. In conclusion, we confirm the potential of ZnuD-bearing OMVs as a component of an effective MenB vaccine.
    Infection and immunity 03/2013; · 4.21 Impact Factor
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    ABSTRACT: The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.
    Environmental Microbiology 03/2013; · 6.24 Impact Factor
  • Jan Grijpstra, Martine P Bos, Jan Tommassen
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    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.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 966:223-37. · 1.29 Impact Factor
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    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.
    PLoS ONE 01/2013; 8(12):e85799. · 3.53 Impact Factor
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    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 exceptional among autotransporters, NalP is a lipoprotein. Here, the authors investigated the role of lipidation in the biogenesis and the 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 the 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.
    Microbiology 12/2012; · 3.06 Impact Factor
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    ABSTRACT: The type II secretion system (T2SS) is a multi-protein assembly spanning the inner and outer-membrane in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization and infection. The exoproteins are secreted across the outer-membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner-membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well-defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of T2SS secretins, and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.
    Journal of Biological Chemistry 11/2012; · 4.65 Impact Factor
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    ABSTRACT: Neisseria meningitidis is a common and usually harmless inhabitant of the mucosa of the human nasopharynx, which, in rare cases, can cross the epithelial barrier and cause meningitis and sepsis. Biofilm formation favours the colonization of the host and the subsequent carrier state. Two different strategies of biofilm formation, either dependent or independent on extracellular DNA (eDNA), have been described for meningococcal strains. Here, we demonstrate that the autotransporter protease NalP, the expression of which is phase variable, affects eDNA-dependent biofilm formation in N. meningitidis. The effect of NalP was found in biofilm formation under static and flow conditions and was dependent on its protease activity. Cleavage of the heparin-binding antigen NhbA and the α-peptide of IgA protease, resulting in the release of positively charged polypeptides from the cell surface, was responsible for the reduction in biofilm formation when NalP is expressed. Both NhbA and the α-peptide of IgA protease were shown to bind DNA. We conclude that NhbA and the α-peptide of IgA protease are implicated in biofilm formation by binding eDNA and that NalP is an important regulator of this process through the proteolysis of these surface-exposed proteins.
    Molecular Microbiology 11/2012; · 5.03 Impact Factor

Publication Stats

3k Citations
534.44 Total Impact Points

Institutions

  • 1996–2014
    • Universiteit Utrecht
      • • Institute of Biomembranes
      • • Division of Microbiology
      • • Bijvoet Institute for Biomolecular Research
      • • Division of Crystal and Structural Chemistry
      • • Centre for Biomembranes and Lipid Enzymology
      Utrecht, Utrecht, Netherlands
  • 2013
    • Erasmus Universiteit Rotterdam
      • Department of Medical Microbiology and Infectious Diseases
      Rotterdam, South Holland, Netherlands
  • 2010
    • University of Amsterdam
      • Department of Molecular Cell Biology
      Amsterdamo, North Holland, Netherlands
  • 2006–2010
    • Netherlands Vaccine Institute (NVI)
      Utrecht, Utrecht, Netherlands
  • 2004–2010
    • Vrije Universiteit Brussel
      • Ultrastructure (ULTR)
      Bruxelles, Brussels Capital Region, Belgium
    • University Medical Center Utrecht
      • Department of Immunology
      Utrecht, Provincie Utrecht, Netherlands
    • Universität Basel
      Bâle, Basel-City, Switzerland
  • 2007
    • Griffith University
      • Institute for Glycomics
      Southport, Queensland, Australia
    • Heinrich-Heine-Universität Düsseldorf
      • Institut für Molekulare Enzymtechnologie (IMET)
      Düsseldorf, North Rhine-Westphalia, Germany
  • 2003
    • Vlaams Instituut voor Biotechnologie
      Gand, Flanders, Belgium
  • 2002
    • National Institute for Public Health and the Environment (RIVM)
      Utrecht, Utrecht, Netherlands