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Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2.

Li Q, Liu H, Du D, Yu Y, Ma C, Jiao F, Yao H, Lu C, Zhang W - Front Cell Infect Microbiol (2015)

Bottom Line: Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay.In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface.Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions.

View Article: PubMed Central - PubMed

Affiliation: Key Lab of Animal Bacteriology, OIE Reference Lab for Swine Streptococcosis, College of Veterinary Medicine, Ministry of Agriculture, Nanjing Agricultural University Nanjing, China.

ABSTRACT
Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5'-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions.

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Determine the binding of the recombinant proteins to LN and FN by ELISA. Microtiter plates were coated with 100 μl of 5 μg/ml purified recombinant proteins and reacted with the indicated concentrations of human LN (A) or FN (B). The negative control was coated with 100 μl of 5 μg/ml casein and incubated with the indicated concentrations of human LN or FN. Binding was evaluated after 2 h. Bound LN or FN was detected with goat anti-rabbit IgG antibody. Data are expressed as the mean ± SD of three independent experiments performed in triplicate.
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Figure 3: Determine the binding of the recombinant proteins to LN and FN by ELISA. Microtiter plates were coated with 100 μl of 5 μg/ml purified recombinant proteins and reacted with the indicated concentrations of human LN (A) or FN (B). The negative control was coated with 100 μl of 5 μg/ml casein and incubated with the indicated concentrations of human LN or FN. Binding was evaluated after 2 h. Bound LN or FN was detected with goat anti-rabbit IgG antibody. Data are expressed as the mean ± SD of three independent experiments performed in triplicate.

Mentions: We selected nine potential LN- or FN-binding proteins for expression and further confirmed by Far-Western blotting and ELISA. After purification by Ni-chelating chromatography, the SDS-PAGE (Figure 2A) and Western blotting analysis (Figure 2B) of the recombinant proteins revealed that the His-tagged fusion proteins were successfully purified. The LN-binding properties of purified recombinant OppA, EF-Tu, enolase, LDH, FBA, KAR, GAPDH, IMPDH, and ABC were verified by Far-Western blot analysis (Figure 2C). A specific response to human FN was also detected for purified recombinant OppA, EF-Tu, enolase, LDH, FBA on the PVDF membrane (Figure 2D). Notably, LN and FN failed to interact with the negative control protein MRP. Qualitatively, the ability of purified recombinant proteins to bind LN or FN was further evaluated by ELISA. The result indicates that the recombinant captured proteins in SS2 interact specifically with LN (Figure 3A) and FN (Figure 3B), and purified recombinant proteins showed concentration dependent binding to human LN or FN. Under similar assay conditions, LN and FN failed to interact with casein, which was served as a negative control. These results provide interesting insights into the role of LN or FN-binding surface proteins of SS2 and clues to the pathogenesis of the SS2 infection.


Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2.

Li Q, Liu H, Du D, Yu Y, Ma C, Jiao F, Yao H, Lu C, Zhang W - Front Cell Infect Microbiol (2015)

Determine the binding of the recombinant proteins to LN and FN by ELISA. Microtiter plates were coated with 100 μl of 5 μg/ml purified recombinant proteins and reacted with the indicated concentrations of human LN (A) or FN (B). The negative control was coated with 100 μl of 5 μg/ml casein and incubated with the indicated concentrations of human LN or FN. Binding was evaluated after 2 h. Bound LN or FN was detected with goat anti-rabbit IgG antibody. Data are expressed as the mean ± SD of three independent experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644805&req=5

Figure 3: Determine the binding of the recombinant proteins to LN and FN by ELISA. Microtiter plates were coated with 100 μl of 5 μg/ml purified recombinant proteins and reacted with the indicated concentrations of human LN (A) or FN (B). The negative control was coated with 100 μl of 5 μg/ml casein and incubated with the indicated concentrations of human LN or FN. Binding was evaluated after 2 h. Bound LN or FN was detected with goat anti-rabbit IgG antibody. Data are expressed as the mean ± SD of three independent experiments performed in triplicate.
Mentions: We selected nine potential LN- or FN-binding proteins for expression and further confirmed by Far-Western blotting and ELISA. After purification by Ni-chelating chromatography, the SDS-PAGE (Figure 2A) and Western blotting analysis (Figure 2B) of the recombinant proteins revealed that the His-tagged fusion proteins were successfully purified. The LN-binding properties of purified recombinant OppA, EF-Tu, enolase, LDH, FBA, KAR, GAPDH, IMPDH, and ABC were verified by Far-Western blot analysis (Figure 2C). A specific response to human FN was also detected for purified recombinant OppA, EF-Tu, enolase, LDH, FBA on the PVDF membrane (Figure 2D). Notably, LN and FN failed to interact with the negative control protein MRP. Qualitatively, the ability of purified recombinant proteins to bind LN or FN was further evaluated by ELISA. The result indicates that the recombinant captured proteins in SS2 interact specifically with LN (Figure 3A) and FN (Figure 3B), and purified recombinant proteins showed concentration dependent binding to human LN or FN. Under similar assay conditions, LN and FN failed to interact with casein, which was served as a negative control. These results provide interesting insights into the role of LN or FN-binding surface proteins of SS2 and clues to the pathogenesis of the SS2 infection.

Bottom Line: Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay.In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface.Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions.

View Article: PubMed Central - PubMed

Affiliation: Key Lab of Animal Bacteriology, OIE Reference Lab for Swine Streptococcosis, College of Veterinary Medicine, Ministry of Agriculture, Nanjing Agricultural University Nanjing, China.

ABSTRACT
Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5'-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions.

Show MeSH
Related in: MedlinePlus