Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis

Jansen, Carmen; Wiese, Andre; Reubsaet, Lieke; Dekker, Niek; Cock, Hans de; Seydel, Ulrich; Tommassen, Jan

doi:10.1016/s0005-2736(00)00155-3

Cited by 61 publications

(73 citation statements)

References 60 publications

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“…The functional unit of porins like MOMP from E. coli and other gram-negative bacteria is usually a homotrimer that migrates with a molecular mass of approximately 65 to 70 kDa on a 10% SDS-PAGE gel (13,27,28,35,54,66). Here, we have shown that the trimer of MOMP under nondenaturing conditions on a 10% SDS-PAGE gel also migrates with an apparent molecular mass of 67 kDa.…”

Section: Discussionmentioning

confidence: 64%

Structural and Functional Analyses of the Major Outer Membrane Protein ofChlamydia trachomatis

et al. 2007

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Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60-and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of ␤-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37°C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a ␤-sheet structure and porin function.Chlamydia strains are ubiquitous pathogens in nature (1,31,33,70). These bacteria have two distinct morphological and functional forms, the elementary bodies (EB) and the reticulate bodies (RB) (33). The EB measure approximately 300 nm in diameter and have a rigid cell membrane, while the RB measure from 1,000 to 1,200 nm in diameter and have a highly pliable membrane. The EB infect eukaryotic cells and become transformed into RB that replicate inside a cytoplasmic inclusion. As a result of replication, the cytoplasmic inclusion progressively enlarges. Following 1 to 3 days of replication, the RB transform again into EB, and cell lysis releases the infectious EB.Unlike other gram-negative bacteria, Chlamydia strains may lack a peptidoglycan layer (50). As a substitute for peptidoglycan, it has been postulated that the 60-kDa (OmcB) and the 12-kDa (OmcA) cysteine-rich proteins (CRPs), and maybe the major outer membrane protein (MOMP) and other membrane components, form a supramolecular structure that provides rigidity to the EB (16, 21, 37, 50, 63). The Chlamydia trachomatis MOMP belongs to a family of proteins found in the outer membranes of gram-negative bacteria that have a molecular mass of approximately 40 kDa and function as porins (40). Porins, such as OmpF and PhoE of Escherichia coli, form homotrimers held together by hydrophobic and electrostatic interactions (40,48). Due to the unusual binding of sodium dodecyl sulfate (SDS), the trimers migrate on 10% SDS-polyacrylamide gel elec...

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Section: Discussionmentioning

confidence: 64%

Structural and Functional Analyses of the Major Outer Membrane Protein ofChlamydia trachomatis

et al. 2007

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“…In single bamB, bamC or bamE mutants, one or a few OMPs are incorrectly assembled, while double mutants have more severe OMP assembly defects or are inviable Sklar et al, 2007;Volokhina et al, 2009;Wu et al, 2005). The N. meningitidis BAM complex contains an additional protein, RmpM (Volokhina et al, 2009), which also associates with the porins PorA and PorB and the TonB-dependent receptors TbpA and LbpA (Jansen et al, 2000;Prinz & Tommassen, 2000). Analysis of Neisseria rmpM mutants suggests that this protein stabilizes OMP complexes rather than participating in OMP assembly (Volokhina et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane β-barrel proteins in Caulobacter crescentus

2010

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The outer membrane of Gram-negative bacteria is an essential compartment containing a specific complement of lipids and proteins that constitute a protective, selective permeability barrier. Outer membrane β-barrel proteins are assembled into the membrane by the essential hetero-oligomeric BAM complex, which contains the lipoprotein BamE. We have identified a homologue of BamE, encoded by CC1365, which is located in the outer membrane of the stalked alpha-proteobacterium Caulobacter crescentus. BamE associates with proteins whose homologues in other bacteria are known to participate in outer membrane protein assembly: BamA (CC1915), BamB (CC1653) and BamD (CC1984). Caulobacter cells lacking BamE grow slowly in rich medium and are hypersensitive to anionic detergents, some antibiotics and heat exposure, which suggest that the membrane integrity of the mutant is compromised. Membranes of the ΔbamE mutant have normal amounts of the outer membrane protein RsaF, a TolC homologue, but are deficient in CpaC*, an aggregated form of the outer membrane secretin for type IV pili. ΔbamE membranes also contain greatly reduced amounts of three TonB-dependent receptors that are abundant in wild-type cells. Cells lacking BamE have short stalks and are delayed in stalk outgrowth during the cell cycle. Based on these findings, we propose that Caulobacter BamE participates in the assembly of outer membrane β-barrel proteins, including one or more substrates required for the initiation of stalk biogenesis.

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“…It was recently reported that a vaccine based on six PorA and five FrpB (FetA) variants should be able to provide protection against a large range of hyperinvasive meningococcal strains [12]. The current work, together with previously developed methods to fold the PorA protein in vitro [13,14], thus enables the development of a protein-based meningococcal subunit vaccine.…”

Section: Discussionmentioning

confidence: 96%

“…Due to their compact conformation the folded monomeric proteins usually migrate more quickly on gels than the heat-denatured form, whereas oligomers migrate more slowly [13]. This so-called heat modifiability of OMPs can be used to monitor their folding in vitro [13,16].…”

Section: In Vitro Folding Of Frpbmentioning

confidence: 99%

Immunogenicity and structural characterisation of an in vitro folded meningococcal siderophore receptor (FrpB, FetA)

Kortekaas

Müller

Ringler

et al. 2006

Microbes and Infection

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The iron-limitation-inducible protein FrpB of Neisseria meningitidis is an outer-membrane-localized siderophore receptor. Because of its abundance and its capacity to elicit bactericidal antibodies, it is considered a vaccine candidate. Bactericidal antibodies against FrpB are, however, type-specific. Hence, an FrpB-based vaccine should comprise several FrpB variants to be capable of providing broad protection. To facilitate the development of a meningococcal subunit vaccine, we have established a procedure to obtain large quantities of the protein in a native-like conformation. The protein was expressed without its signal sequence in Escherichia coli, where it accumulated in inclusion bodies. After in vitro folding, the protein was biochemically, biophysically and biologically characterised. Our results show that in vitro folded FrpB assembles into oligomers, presumably dimers, and that it induces high levels of bactericidal antibodies in laboratory animals.

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Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis

Cited by 61 publications

References 60 publications

Structural and Functional Analyses of the Major Outer Membrane Protein ofChlamydia trachomatis

Structural and Functional Analyses of the Major Outer Membrane Protein ofChlamydia trachomatis

The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane β-barrel proteins in Caulobacter crescentus

Immunogenicity and structural characterisation of an in vitro folded meningococcal siderophore receptor (FrpB, FetA)

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