Limits...
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.

Show MeSH

Related in: MedlinePlus

2-DE gels and Far-Western blot identification of LN- and FN-binding proteins of SS2 CW and EC. The CW and EC proteins were separated in the first dimension by IEF in the pI range of 4–7 and by 12% SDS-PAGE in the second dimension, and then the 2-DE gels transferred to PVDF and incubated with LN or LN. Arrows indicate potential LN- or FN-binding proteins recognized with goat anti-rabbit IgG antibody. (A) 2-DE gel of SS2 CW proteins. (B) Far-Western blot of CW proteins incubated with LN (LN-CW). (C) 2-DE gel of SS2 EC proteins. (D) Far-Western blot of EC proteins incubated with LN (LN-EC). (E) 2-DE gel of SS2 CW proteins. (F) Far-Western blot of CW proteins incubated with FN (FN-CW). (G) 2-DE gel of SS2 EC proteins. (H) Far-Western blot of EC proteins incubated with FN (FN-EC).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4644805&req=5

Figure 1: 2-DE gels and Far-Western blot identification of LN- and FN-binding proteins of SS2 CW and EC. The CW and EC proteins were separated in the first dimension by IEF in the pI range of 4–7 and by 12% SDS-PAGE in the second dimension, and then the 2-DE gels transferred to PVDF and incubated with LN or LN. Arrows indicate potential LN- or FN-binding proteins recognized with goat anti-rabbit IgG antibody. (A) 2-DE gel of SS2 CW proteins. (B) Far-Western blot of CW proteins incubated with LN (LN-CW). (C) 2-DE gel of SS2 EC proteins. (D) Far-Western blot of EC proteins incubated with LN (LN-EC). (E) 2-DE gel of SS2 CW proteins. (F) Far-Western blot of CW proteins incubated with FN (FN-CW). (G) 2-DE gel of SS2 EC proteins. (H) Far-Western blot of EC proteins incubated with FN (FN-EC).

Mentions: Bacterial CW and EC proteins from SS2 were separated by 2D SDS-PAGE over a pH range of 4–7 and transferred to PVDF membranes. Consistent with our previously study, at least 200 Coomassie brilliant blue G250 stained spots were identified on the 2-DE gels (Figures 1A,C). After incubation with LN, 22 LN reactive spots were identified in CW and two in EC (Figures 1B,D). By using the same approach, SS2 CW and EC protein spots in the 2-DE gels were stained with the Coomassie brilliant blue G250 (Figures 1E,G) and five potential SS2 proteins in CW and five in EC that bound to FN were observed (Figures 1F,H). No specific protein spots were observed in the negative control. All the spots were then excised and identified by MALDI-TOF-MS and data were searched against the NCBI sequence database for match. Taken together, proteomic analysis together with Far-Western blotting assays identified 15 potential LN-binding proteins and five potential FN-binding proteins from SS2 surface proteins. All the 15 potential LN-binding proteins and two potential FN-binding proteins were detected for the first time. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) showed specific binding affinities for both LN and FN. The probability score, number of peptide matches, isoelectric point (pI) and molecular weight (MW) enabled confirmed spot identification. All the results are summarized in Table 2.


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)

2-DE gels and Far-Western blot identification of LN- and FN-binding proteins of SS2 CW and EC. The CW and EC proteins were separated in the first dimension by IEF in the pI range of 4–7 and by 12% SDS-PAGE in the second dimension, and then the 2-DE gels transferred to PVDF and incubated with LN or LN. Arrows indicate potential LN- or FN-binding proteins recognized with goat anti-rabbit IgG antibody. (A) 2-DE gel of SS2 CW proteins. (B) Far-Western blot of CW proteins incubated with LN (LN-CW). (C) 2-DE gel of SS2 EC proteins. (D) Far-Western blot of EC proteins incubated with LN (LN-EC). (E) 2-DE gel of SS2 CW proteins. (F) Far-Western blot of CW proteins incubated with FN (FN-CW). (G) 2-DE gel of SS2 EC proteins. (H) Far-Western blot of EC proteins incubated with FN (FN-EC).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: 2-DE gels and Far-Western blot identification of LN- and FN-binding proteins of SS2 CW and EC. The CW and EC proteins were separated in the first dimension by IEF in the pI range of 4–7 and by 12% SDS-PAGE in the second dimension, and then the 2-DE gels transferred to PVDF and incubated with LN or LN. Arrows indicate potential LN- or FN-binding proteins recognized with goat anti-rabbit IgG antibody. (A) 2-DE gel of SS2 CW proteins. (B) Far-Western blot of CW proteins incubated with LN (LN-CW). (C) 2-DE gel of SS2 EC proteins. (D) Far-Western blot of EC proteins incubated with LN (LN-EC). (E) 2-DE gel of SS2 CW proteins. (F) Far-Western blot of CW proteins incubated with FN (FN-CW). (G) 2-DE gel of SS2 EC proteins. (H) Far-Western blot of EC proteins incubated with FN (FN-EC).
Mentions: Bacterial CW and EC proteins from SS2 were separated by 2D SDS-PAGE over a pH range of 4–7 and transferred to PVDF membranes. Consistent with our previously study, at least 200 Coomassie brilliant blue G250 stained spots were identified on the 2-DE gels (Figures 1A,C). After incubation with LN, 22 LN reactive spots were identified in CW and two in EC (Figures 1B,D). By using the same approach, SS2 CW and EC protein spots in the 2-DE gels were stained with the Coomassie brilliant blue G250 (Figures 1E,G) and five potential SS2 proteins in CW and five in EC that bound to FN were observed (Figures 1F,H). No specific protein spots were observed in the negative control. All the spots were then excised and identified by MALDI-TOF-MS and data were searched against the NCBI sequence database for match. Taken together, proteomic analysis together with Far-Western blotting assays identified 15 potential LN-binding proteins and five potential FN-binding proteins from SS2 surface proteins. All the 15 potential LN-binding proteins and two potential FN-binding proteins were detected for the first time. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) showed specific binding affinities for both LN and FN. The probability score, number of peptide matches, isoelectric point (pI) and molecular weight (MW) enabled confirmed spot identification. All the results are summarized in Table 2.

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