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A fibrinogen-binding protein of Staphylococcus epidermidis - PubMed

A fibrinogen-binding protein of Staphylococcus epidermidis

M Nilsson et al. Infect Immun. 1998 Jun.

Abstract

The present study reports on fibrinogen (Fg) binding of Staphylococcus epidermidis. Adhesion of different S. epidermidis strains to immobilized Fg was found to vary significantly between different strains, and the component responsible was found to be proteinaceous in nature. To further characterize the Fg-binding activity, a shotgun phage display library covering the S. epidermidis chromosome was constructed. By affinity selection (panning) against immobilized Fg, a phagemid clone, pSEFG1, was isolated, which harbors an insert with an open reading frame of approximately 1.7 kilobases. Results from binding and inhibition experiments demonstrated that the insert of pSEFG1 encodes a specific Fg-binding protein. Furthermore, affinity-purified protein encoded by pSEFG1 completely inhibited adhesion of S. epidermidis to immobilized Fg. By additional cloning and DNA sequence analyses, the complete gene, termed fbe, was found to consist of an open reading frame of 3,276 nucleotides encoding a protein, called Fbe, with a deduced molecular mass of approximately 119 kDa. With a second phage display library made from another clinical isolate of S. epidermidis, it was possible to localize the Fg-binding region to a 331-amino-acid-long fragment. PCR analysis showed that the fbe gene was found in 40 of 43 clinical isolates of S. epidermidis. The overall organization of Fbe resembles those of other extracellular surface proteins of staphylococci and streptococci. Sequence comparisons with earlier known proteins revealed that this protein is related to an Fg-binding protein of Staphylococcus aureus called clumping factor.

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Figures

FIG. 1
FIG. 1

Bacterial binding to immobilized Fg. Microtiter wells were coated with fibrinogen at the concentration indicated and blocked with BSA. Adherence was allowed for 2 h, microtiter plates were washed and dried, and relative bacterial adherence was determined spectrophotometrically (A405). □, S. aureus Newman; ○, S. epidermidis 2; ▪, S. epidermidis 19; ▵, S. epidermidis 269; •, S. epidermidis 333; ▴ S. epidermidis HB.

FIG. 2
FIG. 2

Schematic presentation of the Fbe protein and alignment of inserts from nine phagemid clones obtained after panning against Fg. The different regions are indicated by S (the signal sequence), A (the Fg-binding region), and R (the highly repetitive region). The insert of the single clone (pSEFG1) originated from strain HB is shown as an open bar, while the eight clones derived from strain 19 are presented by solid lines. The numbers indicate the positions of amino acids (aa) in the Fbe protein as defined in the legend to Fig. 4.

FIG. 3
FIG. 3

(A) Inhibition of binding of phagemid (pSEFG1) particles to microtiter wells coated with Fg. Various concentrations of Fg and HSA were separately mixed with 9 × 105 phagemid particles of pSEFG1. After 1 h of incubation, the samples (200 μl) were transferred to Fg-coated microtiter wells after 3 h of incubation at room temperature. The wells were washed with PBST and subsequently eluted by lowering the pH to 1.9. Aliquots of the eluted phagemid particles were used to infect E. coli TG1 cells and plated on LA plates supplemented with 2% glucose. The resulting number of CFU per milliliter is shown as a function (percentage) of different concentrations of the two soluble plasma proteins added. Points representing the means of duplicates and standard deviations are indicated. (B) SDS-PAGE. Material released by an osmotic shock procedure from an overnight culture of E. coli MC1061 harboring the phagemid pSEFG1 was concentrated by acetone precipitation. E. coli MC1061 cells, with or without the phagemid vector pG8H6, were used as negative controls. After centrifugation, the pellets were resuspended in PBS and boiled in a sample buffer containing SDS and β-mercaptoethanol. Lanes: 1, E. coli MC1061(pSEFG1); 2, E. coli MC1061(pG8H6); 3, E. coli MC1061. Molecular mass markers are indicated. (C) Western blot analysis. After separation by SDS-PAGE, the samples were transferred to NC filters and analyzed for binding of Fg with HRP-labelled anti-Fg antibodies. (D) Inhibition of the adherence of S. epidermidis (strain 19) to immobilized Fg by purified protein of pSEFG1. Microtiter wells coated with Fg were incubated with the indicated amounts of affinity-purified proteins prior to addition of radiolabelled bacteria. After washing, bacterial adherence was determined by scintillation counting. An arrow shows the value in the absence of fusion proteins. □, purified protein expressed by pSEFG1; ▪, purified unrelated histidine fusion protein.

FIG. 4
FIG. 4

Complete nucleotide sequence of the fbe gene from S. epidermidis HB and the deduced amino acid sequence of the encoded protein. A putative ribosomal-binding site (RBS) is underlined, and possible transcription termination hairpin loops are double underlined. The putative signal sequence (S) is followed by the nonrepetitive N-terminal region (A), which harbors the Fg-binding activity. R indicates the highly repetitive region. The amino acid sequence LPDTG, assumed to be involved in cell wall anchoring, is printed in boldface. M indicates the membrane-spanning region, and the translational stop codon is marked with an asterisk.

FIG. 5
FIG. 5

Alignment of the deduced amino acid sequence of the parts in the Fg-binding regions of Fbe and ClfA with the highest similarity. The numbering indicates the amino acid position in Fbe according to Fig. 4 and reference for ClfA. Vertical lines indicate identical amino acids, and dots show similar amino acids. Gaps (indicated by dashes) were introduced to obtain optimal alignment.

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