Publications (99)485.42 Total impact
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Article: Autotransporter secretion: varying on a theme.
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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; · 2.76 Impact Factor -
Article: ZnuD: a potential candidate for a simple and universal Neisseria meningitidis vaccine.
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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 -
Dataset: supplemental figures
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Article: The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases.
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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; · 5.84 Impact Factor -
Article: Assembly of bacterial outer membrane proteins.
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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. -
Article: Lipidation of the autotransporter NalP of Neisseria meningitidis is required for its function in the release of cell-surface-exposed proteins.
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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 -
Dataset: Remans 2010 LolA Paeruginosa
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Article: New insights into the assembly of bacterial secretins: structural studies of the periplasmic domain of XcpQ from Pseudomonas aeruginosa.
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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; · 4.77 Impact Factor -
Article: Involvement of three meningococcal surface-exposed proteins, the heparin-binding protein NhbA, the α-peptide of IgA protease, and the autotransporter protease NalP, in initiation of biofilm formation.
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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; · 5.01 Impact Factor -
Article: Cellular solid-state nuclear magnetic resonance spectroscopy.
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ABSTRACT: Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.Proceedings of the National Academy of Sciences 02/2012; 109(13):4863-8. · 9.68 Impact Factor -
Article: Solid-state NMR spectroscopy on cellular preparations enhanced by dynamic nuclear polarization.
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ABSTRACT: A peek inside: Dynamic nuclear polarization (DNP) enhances the spectroscopic sensitivity of solid-state NMR measurements of uniformly ((13) C,(15) N)- labeled preparations of Escherichia coli cells by more than an order of magnitude (see picture; MW=microwaves, ε=enhancement factor). The major molecular components in the cells can be characterized in this way.Angewandte Chemie International Edition 02/2012; 51(12):2998-3001. · 13.45 Impact Factor -
Article: TMT labelling for the quantitative analysis of adaptive responses in the meningococcal proteome.
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ABSTRACT: In addition to standard gel-based proteomic approaches, gel-free approaches using isobaric label reagents, such as Tandem Mass Tags (TMT), provide a straightforward method for studying adaptations in microbial proteomes to changing environmental conditions. This approach does not have the known difficulties of 2-D gel electrophoresis with proteins of extreme biochemical properties. The workflow described here was designed to study adaptive responses in bacteria and has been applied to study the response of meningococci to iron limitation. The supplemental use of western blotting allows the confirmation of certain changes in protein abundance identified within the TMT study.Methods in molecular biology (Clifton, N.J.) 01/2012; 799:127-41. -
Article: Pseudomonas evades immune recognition of flagellin in both mammals and plants.
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ABSTRACT: The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.PLoS Pathogens 08/2011; 7(8):e1002206. · 9.13 Impact Factor -
Article: The LptD chaperone LptE is not directly involved in lipopolysaccharide transport in Neisseria meningitidis.
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ABSTRACT: The biosynthesis of lipopolysaccharide (LPS) in gram-negative bacteria is well understood, in contrast to the transport to its destination, the outer leaflet of the outer membrane. In Escherichia coli, synthesis and transport of LPS are essential processes. Neisseria meningitidis, conversely, can survive without LPS and tolerates inactivation of genes involved in LPS synthesis and transport. Here, we analyzed whether the LptA, LptB, LptC, LptE, LptF, and LptG proteins, recently implicated in LPS transport in E. coli, function similarly in N. meningitidis. None of the analyzed proteins was essential in N. meningitidis, consistent with their expected roles in LPS transport and additionally demonstrating that they are not required for an essential process such as phospholipid transport. As expected, the absence of most of the Lpt proteins resulted in a severe defect in LPS transport. However, the absence of LptE did not disturb transport of LPS to the cell surface. LptE was found to be associated with LptD, and its absence affected total levels of LptD, suggesting a chaperone-like role for LptE in LptD biogenesis. The absence of a direct role of LptE in LPS transport was substantiated by bioinformatic analyses showing a low conservation of LptE in LPS-producing bacteria. Apparently, the role of LptE in N. meningitidis deviates from that in E. coli, suggesting that the Lpt system does not function in a completely conserved manner in all gram-negative bacteria.Journal of Biological Chemistry 06/2011; 286(33):28688-96. · 4.77 Impact Factor -
Article: Mitochondria can recognize and assemble fragments of a beta-barrel structure.
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ABSTRACT: β-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial β-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full β-barrel structure is required, we expressed in yeast cells the β-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded β-barrel structure to which each monomer is contributing four β-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete β-barrel structure, four β-strands are sufficient for the mitochondria to recognize and assemble a β-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.Molecular biology of the cell 04/2011; 22(10):1638-47. · 5.98 Impact Factor -
Article: Solid-state NMR on a large multidomain integral membrane protein: the outer membrane protein assembly factor BamA.
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ABSTRACT: Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and intrinsic flexibility. We show here that motional-filtered high-resolution solid-state NMR (ssNMR) experiments allow for the observation and structural analysis of very large multidomain membrane proteins that are characterized by different motional time scales. This approach was used to probe the folding of the 790-residue membrane protein BamA, which is the core component of the Escherichia coli outer membrane protein assembly machinery. A combination of dipolar- and scalar-based two-dimensional ssNMR experiments applied to two uniformly (13)C,(15)N-labeled BamA variants revealed characteristic secondary structure elements and distinct dynamics within the BamA transmembrane protein segment and the periplasmic POTRA domains. This approach hence provides a general strategy for collecting atomic-scale structural information on multidomain (membrane) proteins in a native-like environment.Journal of the American Chemical Society 03/2011; 133(12):4175-7. · 9.91 Impact Factor -
Article: Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli.
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ABSTRACT: Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.Molecular Membrane Biology 02/2011; 28(3):158-70. · 2.86 Impact Factor -
Article: Role of the periplasmic chaperones Skp, SurA, and DegQ in outer membrane protein biogenesis in Neisseria meningitidis.
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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.Journal of bacteriology 02/2011; 193(7):1612-21. · 3.94 Impact Factor -
Article: Hydrophobic surface patches on LolA of Pseudomonas aeruginosa are essential for lipoprotein binding.
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ABSTRACT: Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA-lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open beta-barrel covered by alpha helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA-lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches.Journal of Molecular Biology 09/2010; 401(5):921-30. · 4.00 Impact Factor -
Article: Assembly of outer-membrane proteins in bacteria and mitochondria.
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ABSTRACT: The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic alpha-helices, integral outer-membrane proteins (OMPs) form beta-barrels. Similar beta-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these beta-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of beta-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial beta-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.Microbiology 09/2010; 156(Pt 9):2587-96. · 3.06 Impact Factor
Top co-authors
Top Journals
Institutions
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1987–2013
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Universiteit Utrecht
- • Division of Microbiology
- • Bijvoet Institute for Biomolecular Research
- • Institute of Biomembranes
Utrecht, Provincie Utrecht, Netherlands
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2004–2010
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Vrije Universiteit Brussel
Brussels, BRU, Belgium -
Universitair Medisch Centrum Utrecht
- Department of Immunology
Utrecht, Provincie Utrecht, Netherlands
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2009
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Eberhard-Karls-Universität Tübingen
- Interfaculty Institute for Biochemistry
Tübingen, Baden-Wuerttemberg, Germany
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2006
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Netherlands Vaccine Institute (NVI)
Utrecht, Provincie Utrecht, Netherlands
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2002
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RIVM
Utrecht, Provincie Utrecht, Netherlands
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