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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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EF-Tu, enolase, LDH, and FBA are four major SS2 adhesins confirmed by competition inhibition assays. Inhibition of SS2 adhesion to Hep-2 cells by the recombinant proteins (A) and their corresponding polyclonal antibodies (B). Data are expressed as the mean ± SD of three independent experiments. Significant differences are indicated (**P < 0.01; *P < 0.05).
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Figure 5: EF-Tu, enolase, LDH, and FBA are four major SS2 adhesins confirmed by competition inhibition assays. Inhibition of SS2 adhesion to Hep-2 cells by the recombinant proteins (A) and their corresponding polyclonal antibodies (B). Data are expressed as the mean ± SD of three independent experiments. Significant differences are indicated (**P < 0.01; *P < 0.05).

Mentions: To assess the contribution of recombinant proteins to the adhesion of SS2, two inhibition assays were performed at the same time. In the protein inhibition assay, Hep-2 cells were treated with the purified OppA, EF-Tu, enolase, LDH, FBA, IMPDH, and ABC before SS2 adhering to the Hep-2 cells, and BSA was used as negative control. Compared with BSA, recombinant EF-Tu, enolase, LDH, and FBA were found to decrease the adhesion to Hep-2 cells by 27.3, 42.9, 32.7, and 34.7% respectively, and no significant inhibition with other recombinant proteins (Figure 5A). In the antibodies inhibition assay, the polyclonal antibodies against recombinant OppA, EF-Tu, enolase, LDH, and FBA were able to decrease SS2 adherence to Hep-2 cells compared to pre-immune sera (Figure 5B). The level of adherence is expressed as the percent of adherence of ZY05719 without antibody. The results showed that EF-Tu, enolase, LDH, and FBA contribute to the adherence of SS2 to host cells. We consider these proteins to be four major SS2 adhesins.


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)

EF-Tu, enolase, LDH, and FBA are four major SS2 adhesins confirmed by competition inhibition assays. Inhibition of SS2 adhesion to Hep-2 cells by the recombinant proteins (A) and their corresponding polyclonal antibodies (B). Data are expressed as the mean ± SD of three independent experiments. Significant differences are indicated (**P < 0.01; *P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: EF-Tu, enolase, LDH, and FBA are four major SS2 adhesins confirmed by competition inhibition assays. Inhibition of SS2 adhesion to Hep-2 cells by the recombinant proteins (A) and their corresponding polyclonal antibodies (B). Data are expressed as the mean ± SD of three independent experiments. Significant differences are indicated (**P < 0.01; *P < 0.05).
Mentions: To assess the contribution of recombinant proteins to the adhesion of SS2, two inhibition assays were performed at the same time. In the protein inhibition assay, Hep-2 cells were treated with the purified OppA, EF-Tu, enolase, LDH, FBA, IMPDH, and ABC before SS2 adhering to the Hep-2 cells, and BSA was used as negative control. Compared with BSA, recombinant EF-Tu, enolase, LDH, and FBA were found to decrease the adhesion to Hep-2 cells by 27.3, 42.9, 32.7, and 34.7% respectively, and no significant inhibition with other recombinant proteins (Figure 5A). In the antibodies inhibition assay, the polyclonal antibodies against recombinant OppA, EF-Tu, enolase, LDH, and FBA were able to decrease SS2 adherence to Hep-2 cells compared to pre-immune sera (Figure 5B). The level of adherence is expressed as the percent of adherence of ZY05719 without antibody. The results showed that EF-Tu, enolase, LDH, and FBA contribute to the adherence of SS2 to host cells. We consider these proteins to be four major SS2 adhesins.

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