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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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Adherence of recombinant proteins to Hep-2 cells confirmed by an indirect immunofluorescence assay. Hep-2 cells were incubated with purified recombinant OppA, EF-Tu, enolase, LDH, FBA, IMPDH, ABC, or BSA, and then incubated with rabbit antibodies against the corresponding recombinant proteins, stained with goat anti-rabbit IgG-FITC before examined with a fluorescence microscope. The secondary antibody goat anti-rabbit IgG-FITC was used alone to stain Hep-2 cells as a blank control.
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Figure 4: Adherence of recombinant proteins to Hep-2 cells confirmed by an indirect immunofluorescence assay. Hep-2 cells were incubated with purified recombinant OppA, EF-Tu, enolase, LDH, FBA, IMPDH, ABC, or BSA, and then incubated with rabbit antibodies against the corresponding recombinant proteins, stained with goat anti-rabbit IgG-FITC before examined with a fluorescence microscope. The secondary antibody goat anti-rabbit IgG-FITC was used alone to stain Hep-2 cells as a blank control.

Mentions: Indirect immunofluorescence analyses were conducted to test whether recombinant OppA, EF-Tu, enolase, LDH, FBA, IMPDH, and ABC contributed to SS2 adhere to host cells. Among these proteins, the binding activity of SS2 enolase and GAPDH to Hep-2 cells has been proven in the previous study (Wang and Lu, 2007; Zhang et al., 2009a). It has been reported that enolase binds specifically to fibrinogen (Esgleas et al., 2008; Pian et al., 2015), collagen (Esgleas et al., 2008; Zhang et al., 2014a; Pian et al., 2015), fibronectin, and plasminogen (Esgleas et al., 2008; Pian et al., 2015), but its LN-binding property was identified for the first time in this study. The further analysis of enolase may give some insights in the pathogenesis of SS infection. So the protein enolase was used as an internal control and GAPDH was not further studied. Similar to enolase, obvious green signal was observed from the surface of Hep-2 cells pre-incubated with the seven purified recombinant proteins, while no significant green signal was detected from the negative control (BSA) and the blank control (the secondary antibody was used alone to stain Hep-2 cells, Figure 4). The results demonstrate that all seven purified recombinant proteins could interact with Hep-2 cells. Furthermore, we conclude that ECM-binding proteins may contribute to SS2 binding to host cells.


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)

Adherence of recombinant proteins to Hep-2 cells confirmed by an indirect immunofluorescence assay. Hep-2 cells were incubated with purified recombinant OppA, EF-Tu, enolase, LDH, FBA, IMPDH, ABC, or BSA, and then incubated with rabbit antibodies against the corresponding recombinant proteins, stained with goat anti-rabbit IgG-FITC before examined with a fluorescence microscope. The secondary antibody goat anti-rabbit IgG-FITC was used alone to stain Hep-2 cells as a blank control.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Adherence of recombinant proteins to Hep-2 cells confirmed by an indirect immunofluorescence assay. Hep-2 cells were incubated with purified recombinant OppA, EF-Tu, enolase, LDH, FBA, IMPDH, ABC, or BSA, and then incubated with rabbit antibodies against the corresponding recombinant proteins, stained with goat anti-rabbit IgG-FITC before examined with a fluorescence microscope. The secondary antibody goat anti-rabbit IgG-FITC was used alone to stain Hep-2 cells as a blank control.
Mentions: Indirect immunofluorescence analyses were conducted to test whether recombinant OppA, EF-Tu, enolase, LDH, FBA, IMPDH, and ABC contributed to SS2 adhere to host cells. Among these proteins, the binding activity of SS2 enolase and GAPDH to Hep-2 cells has been proven in the previous study (Wang and Lu, 2007; Zhang et al., 2009a). It has been reported that enolase binds specifically to fibrinogen (Esgleas et al., 2008; Pian et al., 2015), collagen (Esgleas et al., 2008; Zhang et al., 2014a; Pian et al., 2015), fibronectin, and plasminogen (Esgleas et al., 2008; Pian et al., 2015), but its LN-binding property was identified for the first time in this study. The further analysis of enolase may give some insights in the pathogenesis of SS infection. So the protein enolase was used as an internal control and GAPDH was not further studied. Similar to enolase, obvious green signal was observed from the surface of Hep-2 cells pre-incubated with the seven purified recombinant proteins, while no significant green signal was detected from the negative control (BSA) and the blank control (the secondary antibody was used alone to stain Hep-2 cells, Figure 4). The results demonstrate that all seven purified recombinant proteins could interact with Hep-2 cells. Furthermore, we conclude that ECM-binding proteins may contribute to SS2 binding to host cells.

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