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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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ELISA reactivities of 14 phage clones (P1–P14) with mAb 2C8 and normal antiserum. ELISA experiments are described in the Materials and Methods. In 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 3: ELISA reactivities of 14 phage clones (P1–P14) with mAb 2C8 and normal antiserum. ELISA experiments are described in the Materials and Methods. In all experiments, absorbance values are means from representative experiments performed in triplicate. Error bars show the range of absorbance values.

Mentions: ELISA was used to confirm that the phage-displayed peptides were specifically recognized by mAb 2C8; normal antiserum was used as a negative control. A phage was considered to be specifically bound by mAb 2C8 when (Aa:Ab) > 0.3, where Aa and Ab correspond to the absorbance of the phage binding to 2C8 mAb and normal antiserum, respectively. Based on these criteria, 14 phage colonies were positive in a sandwich ELISA assay (Fig. 3) and were propagated for inhibition experiments.


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)

ELISA reactivities of 14 phage clones (P1–P14) with mAb 2C8 and normal antiserum. ELISA experiments are described in the Materials and Methods. In 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

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

Figure 3: ELISA reactivities of 14 phage clones (P1–P14) with mAb 2C8 and normal antiserum. ELISA experiments are described in the Materials and Methods. In all experiments, absorbance values are means from representative experiments performed in triplicate. Error bars show the range of absorbance values.
Mentions: ELISA was used to confirm that the phage-displayed peptides were specifically recognized by mAb 2C8; normal antiserum was used as a negative control. A phage was considered to be specifically bound by mAb 2C8 when (Aa:Ab) > 0.3, where Aa and Ab correspond to the absorbance of the phage binding to 2C8 mAb and normal antiserum, respectively. Based on these criteria, 14 phage colonies were positive in a sandwich ELISA assay (Fig. 3) and were propagated for inhibition experiments.

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