Inhibition of biofilm formation by monoclonal antibodies against Staphylococcus epidermidis RP62A accumulation-associated protein - PubMed (original) (raw)

Inhibition of biofilm formation by monoclonal antibodies against Staphylococcus epidermidis RP62A accumulation-associated protein

Daqian Sun et al. Clin Diagn Lab Immunol. 2005 Jan.

Abstract

Staphylococcus epidermidis expresses a 140-kDa cell wall-bound protein accumulation-associated protein (AAP) to adhere to and accumulate as a biofilm on a surface. Potentially blocking AAP with a monoclonal antibody (MAb) could reduce or eliminate S. epidermidis bacterial colonization of biomedical devices. Here, we report on our efforts to (i) isolate AAP, (ii) generate MAbs against AAP, and (iii) determine the efficacy of MAbs to inhibit S. epidermidis biofilm formation. An M7 S. epidermidis mutant, reportedly deficient in AAP expression, was used as a negative control. Postinoculation murine sera, containing polyclonal antibodies against AAP, were able to reduce S. epidermidis biofilm formation by 54%. Select MAbs against AAP were able to reduce S. epidermidis by no more than 66%. Two MAb mixtures, 12C6/12A1 and 3C1/12A1, reduced S. epidermidis accumulation up to 79 and 87%, respectively, significantly more than individual MAbs. Contrary to a previous report, biofilm-deficient S. epidermidis mutant M7 expressed a 200-kDa protein on its cell wall that specifically bound AAP MAbs. Peptide characterization of this M7 protein by microcapillary reversed-phase high-pressure liquid chromatography-nanoelectrospray tandem mass spectrometry resulted in 53% homology with AAP. Ongoing studies will elucidate the dynamic expression of AAP and the M7 200-kDa protein in order to define their roles in biofilm formation.

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Figures

FIG. 1.

FIG. 1.

Coomassie blue-stained 7.5% Tris-HCl polyacrylamide gel. Lanes: A, extracellular proteins of S. epidermidis RP62A (sessile growth); B, extracellular proteins of S. epidermidis M7 (sessile growth); C, cell wall protein of S. epidermidis RP62A (sessile growth); D, cell wall protein of S. epidermidis M7 (sessile growth). Arrow, 200-kDa protein; E, molecular weight markers.

FIG. 2.

FIG. 2.

Indirect protein ELISA of MAb 12C6 with 140-kDa AAP. A 96-well ELISA plate was coated with AAP. MAb 12C6 was added to each well and incubated for 3 h at 37°C. Bound MAb were detected with alkaline phosphatase-conjugated goat anti-mouse IgG. □, MAb 12C6; ▪, MAb 1934 (an irrelevant MAb against fibronectin).

FIG. 3.

FIG. 3.

Competitive ELISA. Each well of a 96-well plate was coated with 200 ng of AAP. Various concentrations of AAP (4.8 ng/ml to 20 μg/ml were mixed with 100 ng of MAb 12C6/ml. The mixtures were added to each well, and the plate was incubated at 37°C for 3 h. Bound MAb were detected with alkaline phosphatase-conjugated goat anti-mouse IgG and PNPP. MAb binding inhibition is defined in the text.

FIG. 4.

FIG. 4.

Western blot of extracellular proteins and cell wall protein from S. epidermidis RP62A and M7 probed with MAb 12C6. Lane A, extracellular proteins from RP62A; lane B, cell wall protein of RP62A; lane C, extracellular proteins from M7; lane D, cell wall protein from M7.

FIG. 5.

FIG. 5.

Western blot of extracellular proteins and cell wall protein from S. epidermidis RP62A and M7 probed with F(ab′)2 fragments of MAb 12C6. Lane A, extracellular proteins from RP62A; lane B, cell wall protein of RP62A; lane C, extracellular proteins from M7; lane D, cell wall protein from M7.

FIG. 6.

FIG. 6.

S. epidermidis RP62A biofilm inhibition by MAb 12C6 in a 96-well polystyrene plate. Bacteria were suspended in chemically defined medium containing the indicated concentrations of MAb and incubated for 2 h at 4°C and then overnight at 37°C. Formation of the biofilm was measured with safranin O stain. □, F(ab′)2; ▪, MAb 12C6. Biofilm inhibition is defined in the text.

FIG. 7.

FIG. 7.

Double-sandwich ELISA. Each well of a 96-well ELISA plate was exposed to 100 ng of purified capture MAb at room temperature overnight. AAP at 1 μg/ml was added to each well and incubated for 3 h at 37°C. After three washes with PBST, a detection antibody labeled with biotin was added to an appropriate well and allowed to react with the bound AAP for 2 h at 37°C. Bound biotin-labeled MAb were detected with streptavidin-alkaline phosphatase and PNPP. Other data for an AAP concentration range from 1 to 250 ng/ml are available.

FIG. 8.

FIG. 8.

S. epidermidis RP62A biofilm inhibition in a 96-well polystyrene plate. Bacteria were suspended in chemically defined medium containing the indicated concentration of MAb and incubated for 2 h at 4°C and then overnight at 37°C. Formation of the biofilm was measured with safranin O stain. (a) Biofilm inhibition by individual MAbs. (b) Biofilm inhibition by MAb mixtures.

References

    1. Abiko, Y. 2000. Passive immunization against dental caries and periodontal disease: development of recombinant and human monoclonal antibodies. Crit. Rev. Oral Biol. Med. 11**:**140-158. - PubMed
    1. Azghani, A. O., S. Idell, M. Bains, and R. E. Hancock. 2002. Pseudomonas aeruginosa outer membrane protein F is an adhesin in bacterial binding to lung epithelial cells in culture. Microb. Pathog. 33**:**109-114. - PubMed
    1. Booth, V., F. P. Ashley, and T. Lehner. 1996. Passive immunization with monoclonal antibodies against Porphyromonas gingivalis in patients with periodontitis. Infect. Immun. 64**:**422-427. - PMC - PubMed
    1. Brown, J. C., and M. E. Koshland. 1977. Evidence for a long-range conformational change induced by antigen binding to IgM antibody. Proc. Natl. Acad. Sci. USA 74**:**5682-5686. - PMC - PubMed
    1. Cheung, A. L., and V. A. Fischetti. 1988. Variation in the expression of cell wall proteins of Staphylococcus aureus grown on solid and liquid media. Infect. Immun. 56**:**1061-1065. - PMC - PubMed

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