Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

Achtman, Mark; Neibert, Michael; Crowe, Brian A.; Strittmatter, Wolfgang; Kušećek, Barica; Weyse, E; Walsh, Martin J.; Slawig, B; Morelli, Giovanna; Moll, A

doi:10.1084/jem.168.2.507

Cited by 158 publications

(128 citation statements)

References 29 publications

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“…General methods SDS-PAGE and Western blotting were performed using 11% polyacrylamide gels (Achtman et al, 1988), supplemented with 3 M urea as required. Isolation of plasmid DNA, restriction endonuclease cleavage, alkaline phosphatase treatment, DNA phosphorylation, ligation, and transformation of E. coli were performed according to standard methods (Maniatis et al, 1982).…”

Section: Methodsmentioning

confidence: 99%

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Merker

Tommassen

Kušećek

et al. 1997

Molecular Microbiology

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SummaryA two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surfaceexposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (ٌ) from Semliki Forest virus were inserted into Bgl II restriction sites created by site-directed mutagenesis. The ٌ epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while ٌ epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

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Section: Methodsmentioning

confidence: 99%

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Merker

Tommassen

Kušećek

et al. 1997

Molecular Microbiology

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“…Con®rmation of Opa protein expression was veri®ed by SDS±PAGE and immunoblot analysis of total bacterial extracts obtained from cultures used for infection. The monoclonal antibody 4B12C11 (Achtman et al, 1988), generously provided by Dr Mark Achtman, Berlin, Germany, was used for the detection of Opa proteins.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding

et al. 1999

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SummaryThe carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEA-CAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa 52 involves residues 27±42, which are predicted to form b-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27±29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEA-CAM6 receptors that are bound by CEACAM1/CEA-CAM5-speci®c Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC 8 face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.

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“…Bacteria were grown on GCB agar (Difco) with Kellogg's supplements I and II, and maintained at 37 uC, 5 % CO 2 . PilT expression, piliation and Opa status of all strains were monitored by light microscopy of colony morphology, immunoblotting with polyclonal antibodies against PilT (gift of K. T. Forest), and mAbs against pilin (Merz & So, 1997) and Opa (Achtman et al, 1988).…”

Section: Methodsmentioning

confidence: 99%

Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex

et al. 2009

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Early in infection, Neisseria gonorrhoeae can be observed to attach to the epithelial cell surface as microcolonies and induce dramatic changes to the host cell cortex. We tested the hypothesis that type IV pili (Tfp) retraction plays a role in the ultrastructure of both the host cell cortex and the bacterial microcolony. Using serial ultrathin sectioning, transmission electron microscopy and 3D reconstruction of serial 2D images, we have obtained what we believe to be the first 3D reconstructions of the N. gonorrhoeae-host cell interface, and determined the architecture of infected cell microvilli as well as the attached microcolony. Tfp connect both wild-type (wt) and Tfp retraction-deficient bacteria with each other, and with the host cell membrane. Tfp fibres and microvilli form a lattice in the wt microcolony and at its periphery. Wt microcolonies induce microvilli formation and increases of surface area, leading to an approximately ninefold increase in the surface area of the host cell membrane at the site of attachment. In contrast, Tfp retractiondeficient microcolonies do not affect these parameters. Wt microcolonies had a symmetrical, dome-shaped structure with a circular 'footprint', while Tfp retraction-deficient microcolonies were notably less symmetrical. These findings support a major role for Tfp retraction in microvilli and microcolony architecture. They are consistent with the biophysical attributes of Tfp and the effects of Tfp retraction on epithelial cell signalling. INTRODUCTIONNeisseria gonorrhoeae (the gonococcus, or GC) remains a significant public health concern. The Gram-negative diplococcus causes a million cases of gonorrhoea annually in Western Europe and North America, and over 62 million cases worldwide (World Health Organization). Humans are the only known reservoir for GC. The exquisite tropism of GC for man and the ability of GC to establish a carrier state (Turner et al., 2002) reflect a highly evolved relationship between pathogen and host.Gonococcal attachment is promoted by type IV pili (Tfp), peritrichous fibres that are 6 nm in width and up to several micrometres in length. The Tfp filament is composed of helical polymers of pilin subunits (Craig et al., 2006;Parge et al., 1995) and minor proteins (Carbonnelle et al., 2005;Winther-Larsen et al., 2005). Tfp participate in important biological processes such as DNA uptake/genetic exchange, twitching motility, attachment, and host cell signalling (Mattick, 2002;. These activities require, or are enhanced by, the retraction of Tfp fibres (Wolfgang et al., 2000).Gonococcal Tfp retraction is an activity that requires PilT, the Tfp retraction motor subunit (Wolfgang et al., 1998). Retracting Tfp generate strong 'pull' forces, ranging from 50 picoNewtons (pN) to 1 nanoNewton (nN) (Biais et al., 2008;Opitz et al., 2009). Repeated cycles of Tfp extension, substrate attachment and Tfp retraction allow GC to crawl over surfaces and aggregate into microcolonies. Tfp retraction from bacteria in a microcolony allows the community to craw...

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Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

Cited by 158 publications

References 29 publications

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding

Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex

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