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Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

Śmiga M, Bielecki M, Olczak M, Smalley JW, Olczak T - PLoS ONE (2015)

Bottom Line: First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo.No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected.The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.

ABSTRACT
Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

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Presentation of P. gingivalis HmuY epitopes in amino acid sequence (A) and three-dimensional protein structure (B).Epitopes analyzed are indicated in red.
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pone.0117508.g001: Presentation of P. gingivalis HmuY epitopes in amino acid sequence (A) and three-dimensional protein structure (B).Epitopes analyzed are indicated in red.

Mentions: This study extends our previously published data and presents characterization of the HmuY protein with regard to its future application to determine specific anti-P. gingivalis antibodies in serum. The best antigen targets are bacterial components, which are constitutively expressed and secreted from the bacterium or exposed on the pathogen’s surface. The use of whole-sonicate antigens, which encompass many of the P. gingivalis proteins [53,54], would increase the possibility of detecting responses, but also increase the potential for false positive results because of cross-reacting antibodies to components of other bacteria. Therefore, to detect specific anti-P. gingivalis antibodies which do not cross react with components produced by other bacteria, we employed purified HmuY protein or its selected epitopes as antigens (Fig. 1). Since other periodontopathogens produce HmuY homologs, we aimed to characterize responses of antibodies raised against HmuY protein or its epitopes to the most homologous proteins from P. intermedia and T. forsythia. Taking into account the amino acid sequences and the theoretically modeled three-dimensional protein structures, we found the two closest HmuY homologs in P. intermedia 17, termed here PinA and PinO, and one HmuY homolog from T. forsythia ATCC 43037, termed here Tfo (Fig. 2). For the latter, we identified corrected DNA (Fig. 3A) and amino acid (Fig. 3B) sequences (EMBL accession number LN624459). It appears that previously in databases a nucleotide sequence possessing a 6-bp insertion and nucleotide substitutions has been deposited. In contrast, the amino acid sequence of Tfo identified in our laboratory contains both a two-amino-acid deletion and substitutions of nine amino acids (Fig. 3C). We did not identify the HmuY homolog in T. denticola, the third member of the "red complex" of periodontopathogens. In this study we overexpressed all proteins and established purification procedures for the PinA, PinO and Tfo proteins.


Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

Śmiga M, Bielecki M, Olczak M, Smalley JW, Olczak T - PLoS ONE (2015)

Presentation of P. gingivalis HmuY epitopes in amino acid sequence (A) and three-dimensional protein structure (B).Epitopes analyzed are indicated in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320075&req=5

pone.0117508.g001: Presentation of P. gingivalis HmuY epitopes in amino acid sequence (A) and three-dimensional protein structure (B).Epitopes analyzed are indicated in red.
Mentions: This study extends our previously published data and presents characterization of the HmuY protein with regard to its future application to determine specific anti-P. gingivalis antibodies in serum. The best antigen targets are bacterial components, which are constitutively expressed and secreted from the bacterium or exposed on the pathogen’s surface. The use of whole-sonicate antigens, which encompass many of the P. gingivalis proteins [53,54], would increase the possibility of detecting responses, but also increase the potential for false positive results because of cross-reacting antibodies to components of other bacteria. Therefore, to detect specific anti-P. gingivalis antibodies which do not cross react with components produced by other bacteria, we employed purified HmuY protein or its selected epitopes as antigens (Fig. 1). Since other periodontopathogens produce HmuY homologs, we aimed to characterize responses of antibodies raised against HmuY protein or its epitopes to the most homologous proteins from P. intermedia and T. forsythia. Taking into account the amino acid sequences and the theoretically modeled three-dimensional protein structures, we found the two closest HmuY homologs in P. intermedia 17, termed here PinA and PinO, and one HmuY homolog from T. forsythia ATCC 43037, termed here Tfo (Fig. 2). For the latter, we identified corrected DNA (Fig. 3A) and amino acid (Fig. 3B) sequences (EMBL accession number LN624459). It appears that previously in databases a nucleotide sequence possessing a 6-bp insertion and nucleotide substitutions has been deposited. In contrast, the amino acid sequence of Tfo identified in our laboratory contains both a two-amino-acid deletion and substitutions of nine amino acids (Fig. 3C). We did not identify the HmuY homolog in T. denticola, the third member of the "red complex" of periodontopathogens. In this study we overexpressed all proteins and established purification procedures for the PinA, PinO and Tfo proteins.

Bottom Line: First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo.No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected.The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.

ABSTRACT
Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

Show MeSH
Related in: MedlinePlus