The localisation of phoE and ?-galactosidase antigens in wild type and phoE/lacZ hybrid

Verkleij, A.J.; Leunissen, J. L. M.; Damme-Jongsten, M. van; Tommassen, Jan; Lugtenberg, Ben

doi:10.1016/0309-1651(84)90026-2

Cited by 2 publications

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“…One of these proteins is the pore-forming protein PhoE, which is synthesized in the cytoplasm, transported to and incorporated in the outer membrane as a functional trimer. This processing has been subject of study in our laboratory for several years, by means of immunocytochemical labelling at the EM level Leunissen et al, 1984a). In ultrathin cryosections of E .…”

Section: Introductionmentioning

confidence: 99%

“…In ultrathin cryosections of E . coli K-12 cells the PhoE pore protein complex has been localized in the outer membrane by the use of monoclonal antibodies against PhoE trimer in combination with protein A-gold probes (Leunissen et al, 1984a;Tommassen et al, 1985). Cryo-0 1986 The Royal Microscopical Society ultramicrotomy has proven to be an excellent method to localize intracellular-and cell-surface located antigens (Tokuyasu, 1980;Geuze et al, 1983;Boonstra et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

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Steric hindrance in immunolabelling

Voorhout

Leunissen‐Bijvelt

Leunissen

et al. 1986

Journal of Microscopy

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In this paper we describe immunocytochemical detection of PhoE pore protein in the outer membrane of E . coli K-12 cells in dependence of a variety of labelling approaches.Immuno-gold labelling on ultrathin cryosections showed a uniform, dense labelling of the outer membrane of all cells. However, using immunofluorescence, whole-mount or freeze-etch labelling methods, labelling was limited to less than 5% of the cell population.In order to understand this phenomenon, immunoincubated cells exhibiting less than 5% labelling were processed for cryo-ultramicrotomy. Reincubation of the sections with antibody and probe resulted in labelling of all of the cells.If an E . coli Gal-U mutant strain, defective in the lipopolysaccharide (LPS) carbohydrate chain length, was used, each approach rendered 100% labelling. From these results it is concluded that the antigenic sites of the PhoE pore protein are not accessible in intact 'wild-type' cells due to steric hindrance caused by the LPS carbohydrate chains. In cryosections this steric hindrance is partly precluded since antigenic determinants are exposed at the section surface in transversely cut membranes.Our results emphasize the necessity to compare results obtained by means of several, basically different approaches.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Steric hindrance in immunolabelling

Voorhout

Leunissen‐Bijvelt

Leunissen

et al. 1986

Journal of Microscopy

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“…After being washed with phosphate-buffered saline (PBS), the bacteria were resuspended and fixed for 1 h in PBS containing 2% paraformaldehyde and 0.25% glutaraldehyde. Subsequent cryo-ultramicrotomy and immunogold labeling were performed as described previously (11,20), with a goat anti-mouse 1-nmdiameter gold-bead antibody (Aurion, Wageningen, The Netherlands). After the labeling procedure, a Danscher silver enhancement (20 to 25 min) (7), washing with milliQ water (three times for 2 min each), and negative staining with uranyl acetate (saturated, 2 min) were done.…”

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confidence: 99%

Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis

Veenstra¹,

Cremers²,

Dijk³

et al. 1996

J Bacteriol

146

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Coagulase-negative staphylococci have emerged as important pathogens in infections associated with intravascular devices. Microbial adherence to biomaterial surfaces is a crucial step in the pathogenesis of these infections. Staphylococcal surface proteins (herein referred to as SSP-1 and SSP-2) are involved in the attachment of Staphylococcus epidermidis 354 to polystyrene. In the present study we show that the adhesin protrudes from the cell surface as a fimbria-like polymer. Furthermore, in vitro proteolytic cleavage of SSP-1 produces an SSP-2-like protein which coincides with a loss of adhesive function. SSP-1 expression is downregulated in a phenotypical variant of S. epidermidis 354 whereas SSP-2 expression is not. These results could suggest that proteolytic cleavage is a key to the regulation of the adhesive state of S. epidermidis in vivo.Over the last 2 decades coagulase-negative staphylococci (CoNS), particularly strains of Staphylococcus epidermidis, have emerged as important pathogens in association with foreign bodies, e.g., intravascular devices. Especially in immunocompromised patients, such as cancer patients and premature neonates, catheter-related sepsis due to CoNS may be a lifethreatening complication (1, 2, 5, 12, 13). For many microorganisms, adhesion to host surfaces is a crucial step in the pathogenic process and a prerequisite for colonization of these surfaces (3). It has been demonstrated that CoNS adhere avidly to a variety of biomaterials such as Teflon and polystyrene (6,8,16,19). The initial phase of attachment was found to be correlated with surface hydrophobicity. Both hydrophobicity and adherence were reduced by proteases, such as pepsin, suggesting that hydrophobic cell surface proteins may play an important role in the initial attachment to biomaterials (8,16). In a previous study (19), we identified a proteinaceous antigen of S. epidermidis 354 that apparently is involved in adherence to polystyrene. Evidence was obtained that this antigen, herein referred to as staphylococcal surface proteins (SSP-1 and SSP-2), is present on fimbria-like appendages protruding from the bacterial cell surface. A monoclonal antibody specific for SSP-1 and SSP-2 was shown to be specific and as effective as proteases in preventing this staphylococcal strain from adhering to polystyrene (19). This strain thus provides a model system to study the adherence of CoNS to intravascular devices. In the present study we present data concerning the ultrastructural organization of the staphylococcal fimbria-like appendages, the structural and functional relationship between the antigenically related SSP-1 and SSP-2 proteins, and aspects of adhesion regulation in vivo. For cryo-ultramicrotomy and immunogold-labeling purposes, staphylococci, grown on blood agar plates, were inoculated in nutrient broth (Difco) and cultured overnight at 37ЊC. After being washed with phosphate-buffered saline (PBS), the bacteria were resuspended and fixed for 1 h in PBS containing 2% paraformaldehyde and 0.25% glutaraldehyde. ...

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The localisation of phoE and ?-galactosidase antigens in wild type and phoE/lacZ hybrid

Cited by 2 publications

References 0 publications

Steric hindrance in immunolabelling

Steric hindrance in immunolabelling

Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis

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