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Determination of the mimic epitope of the M-like protein adhesin in swine Streptococcus equi subsp. zooepidemicus.

Fan H, Wang Y, Tang F, Lu C - BMC Microbiol. (2008)

Bottom Line: Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner.Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP.The motif XSLSRX was highly conserved among six of the ten clones.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Veterinary Medcine, Nanjing Agricultural University, Nanjing 210095, PR China. fhj@njau.edu.cn

ABSTRACT

Background: The M-like protein, also known as SzP, is expressed on the surface of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Previous studies demonstrated that SzP is similar to M protein of group A Streptococcus in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of S. zooepidemicus also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells.

Methods: The gene encoding SzP was expressed in E. coli, and the purified recombinant SzP (rSzP) was recognized by rabbit anti-S. zooepidemicus antibodies using immunoblot. Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner. We employed a random 12-peptide phage display library for screening of immunodominant mimics of the SzP, which were recognized by an anti-SzP specific monoclonal antibody (mAb 2C8). Initial positive phage clones were identified by ELISA, followed by assays to determine the adherence-inhibiting ability of the peptide.

Results: Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP. The motif XSLSRX was highly conserved among six of the ten clones.

Conclusion: Collectively, our findings suggest that the motif XSLSRX may represent an immunodominant mimic epitope of the SzP of S. zooepidemicus strain ATCC 35246, and that the same epitope may be used to mediate SzP binding to HEp-2 cells.

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Characterization of purified rSzP protein binding to HEp-2 cells by ELISA. (A) Binding of biotinylated S. zooepidemicus whole cells, rSzP protein, and a fetuin control to HEp-2 monolayers. (B) Dose-dependent binding of rSzP to HEp-2 cells. (C) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with rabbit polyclonal anti-SzP antibodies. (D) No inhibition of HEp-2 binding with control rabbit serum. (E) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with monoclonal antibodies against SzP. For all experiments, absorbance values are means from representative experiments performed in triplicate. Error bars show the range of absorbance values.
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Figure 2: Characterization of purified rSzP protein binding to HEp-2 cells by ELISA. (A) Binding of biotinylated S. zooepidemicus whole cells, rSzP protein, and a fetuin control to HEp-2 monolayers. (B) Dose-dependent binding of rSzP to HEp-2 cells. (C) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with rabbit polyclonal anti-SzP antibodies. (D) No inhibition of HEp-2 binding with control rabbit serum. (E) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with monoclonal antibodies against SzP. For all experiments, absorbance values are means from representative experiments performed in triplicate. Error bars show the range of absorbance values.

Mentions: The binding of purified rSzP to HEp-2 monolayers was assessed by ELISA. Purified rSzP exhibited significant, dose-dependent binding to HEp-2 cells, whereas binding of the negative control, fetuin, was negligible (Fig. 2A and 2B). Adherence inhibition assays were used to determine the ability of anti-SzP antibodies to inhibit adherence of S. zooepidemicus to HEp-2 cells. Biotinylated S. zooepidemicus intact cells were first pretreated with the anti- SzP polyclonal/monoclonal antibodies and were then tested for the ability to bind HEp-2 monolayers. Pretreatment of biotinylated S. zooepidemicus with increasing amounts of anti- SzP polyclonal antibodies resulted in a significant (P < 0.01) dose-dependent inhibition of bacterial adherence (23%, 48%, and 58% reduction at 1:1,000, 1:100, and 1:10 dilutions, respectively) (Fig. 2C). No significant difference (P > 0.05) was observed in the adherence of normal serum-treated bacteria to HEp-2 cells (Fig. 2D). Out of the 12 monoclonal antibodies,only 2C8 was able to inhibit the adherence of S. zooepidemicus to HEp-2 cells (Fig. 2E).


Determination of the mimic epitope of the M-like protein adhesin in swine Streptococcus equi subsp. zooepidemicus.

Fan H, Wang Y, Tang F, Lu C - BMC Microbiol. (2008)

Characterization of purified rSzP protein binding to HEp-2 cells by ELISA. (A) Binding of biotinylated S. zooepidemicus whole cells, rSzP protein, and a fetuin control to HEp-2 monolayers. (B) Dose-dependent binding of rSzP to HEp-2 cells. (C) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with rabbit polyclonal anti-SzP antibodies. (D) No inhibition of HEp-2 binding with control rabbit serum. (E) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with monoclonal antibodies against SzP. For all experiments, absorbance values are means from representative experiments performed in triplicate. Error bars show the range of absorbance values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2567331&req=5

Figure 2: Characterization of purified rSzP protein binding to HEp-2 cells by ELISA. (A) Binding of biotinylated S. zooepidemicus whole cells, rSzP protein, and a fetuin control to HEp-2 monolayers. (B) Dose-dependent binding of rSzP to HEp-2 cells. (C) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with rabbit polyclonal anti-SzP antibodies. (D) No inhibition of HEp-2 binding with control rabbit serum. (E) Inhibition of S. zooepidemicus binding to HEp-2 monolayers following pretreatment of bacteria with monoclonal antibodies against SzP. For all experiments, absorbance values are means from representative experiments performed in triplicate. Error bars show the range of absorbance values.
Mentions: The binding of purified rSzP to HEp-2 monolayers was assessed by ELISA. Purified rSzP exhibited significant, dose-dependent binding to HEp-2 cells, whereas binding of the negative control, fetuin, was negligible (Fig. 2A and 2B). Adherence inhibition assays were used to determine the ability of anti-SzP antibodies to inhibit adherence of S. zooepidemicus to HEp-2 cells. Biotinylated S. zooepidemicus intact cells were first pretreated with the anti- SzP polyclonal/monoclonal antibodies and were then tested for the ability to bind HEp-2 monolayers. Pretreatment of biotinylated S. zooepidemicus with increasing amounts of anti- SzP polyclonal antibodies resulted in a significant (P < 0.01) dose-dependent inhibition of bacterial adherence (23%, 48%, and 58% reduction at 1:1,000, 1:100, and 1:10 dilutions, respectively) (Fig. 2C). No significant difference (P > 0.05) was observed in the adherence of normal serum-treated bacteria to HEp-2 cells (Fig. 2D). Out of the 12 monoclonal antibodies,only 2C8 was able to inhibit the adherence of S. zooepidemicus to HEp-2 cells (Fig. 2E).

Bottom Line: Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner.Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP.The motif XSLSRX was highly conserved among six of the ten clones.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Veterinary Medcine, Nanjing Agricultural University, Nanjing 210095, PR China. fhj@njau.edu.cn

ABSTRACT

Background: The M-like protein, also known as SzP, is expressed on the surface of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Previous studies demonstrated that SzP is similar to M protein of group A Streptococcus in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of S. zooepidemicus also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells.

Methods: The gene encoding SzP was expressed in E. coli, and the purified recombinant SzP (rSzP) was recognized by rabbit anti-S. zooepidemicus antibodies using immunoblot. Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner. We employed a random 12-peptide phage display library for screening of immunodominant mimics of the SzP, which were recognized by an anti-SzP specific monoclonal antibody (mAb 2C8). Initial positive phage clones were identified by ELISA, followed by assays to determine the adherence-inhibiting ability of the peptide.

Results: Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP. The motif XSLSRX was highly conserved among six of the ten clones.

Conclusion: Collectively, our findings suggest that the motif XSLSRX may represent an immunodominant mimic epitope of the SzP of S. zooepidemicus strain ATCC 35246, and that the same epitope may be used to mediate SzP binding to HEp-2 cells.

Show MeSH
Related in: MedlinePlus