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The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen.

Bober M, Mörgelin M, Olin AI, von Pawel-Rammingen U, Collin M - PLoS ONE (2011)

Bottom Line: Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance.In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface.This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection Medicine, Department of Clinical Sciences, Biomedical Center, Lund University, Lund, Sweden. marta.bober@med.lu.se

ABSTRACT
Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.

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Slr gene disruption, mutant strain analysis and protein localization by electron microscopy.A: Schematic representation of the mutagenesis strategy used to disrupt slr in S. pyogenes strain AP1. B: SDS-PAGE and Western blot analysis, using antibodies against Slr and M1, of bacterial cell extracts of the mutant strains MB1 and MC25, and wild type bacteria AP1. C: Wild type strain AP1, mutant strain MB1 and mutant strain MC25 incubated with gold labeled F(ab')2 fragments of anti-Slr (small gold particles) and anti-M1 (big gold particles) IgG. The dots represent binding of the F(ab')2 fragments to the Slr and M1 protein on the bacterial surface. Arrowheads without arrows indicate binding of anti-Slr antibodies, and complete arrows indicate binding of anti-M1 antibodies in all three panels. Scale bar  = 100 nm.
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pone-0020345-g003: Slr gene disruption, mutant strain analysis and protein localization by electron microscopy.A: Schematic representation of the mutagenesis strategy used to disrupt slr in S. pyogenes strain AP1. B: SDS-PAGE and Western blot analysis, using antibodies against Slr and M1, of bacterial cell extracts of the mutant strains MB1 and MC25, and wild type bacteria AP1. C: Wild type strain AP1, mutant strain MB1 and mutant strain MC25 incubated with gold labeled F(ab')2 fragments of anti-Slr (small gold particles) and anti-M1 (big gold particles) IgG. The dots represent binding of the F(ab')2 fragments to the Slr and M1 protein on the bacterial surface. Arrowheads without arrows indicate binding of anti-Slr antibodies, and complete arrows indicate binding of anti-M1 antibodies in all three panels. Scale bar  = 100 nm.

Mentions: The Slr protein was expressed in E. coli using the GST Gene Fusion system and was used in several in vitro experiments (See Material and Methods). GST-Slr and Slr were separated on a SDS-PAGE for size and purity control. The GST-Slr has an apparent size of 110 kDa and Slr of approximately 85 kDa (Figure 2C). To confirm that Slr is a lipoprotein, a globomycin inhibition assay was performed. The maturation of lipoproteins is governed by signal peptidase II that removes signal peptides in the NH2-terminal signal sequence. This step is inhibited by globomycin, resulting in a surplus of signal peptides and thus an increased molecular size (Figure 2D), confirming that Slr is a lipoprotein. To investigate the role of Slr on the surface of S. pyogenes, we constructed a Slr mutant by single crossover mutagenesis of the AP1 strain of M1 serotype. A 505 bp promotorless fragment of the slr gene was cloned into multiple cloning site I of pFW13, resulting in vector pMB01 (Figure 3A). One mutant strain denoted MB1 was chosen for further analysis. Correct insertion of pMB01 was confirmed by PCR using gene- and plasmid specific primers (Figure 3A, primer 1–4, data not shown). To confirm slr disruption in MB1 and the Slr and M1 expression in AP1 and MC25, protein extracts from AP1, MB1 and MC25 cell pellets were analyzed by immunoblotting using anti-Slr and anti-M1 antibodies. A strong signal was detected in the AP1 pellet for both Slr and M1 protein. The only detectable signal in MB1 was for the M1 protein while in MC25 only Slr expression was detected (Figure 3B). Further mutant strain analysis and surface protein localization was done using gold labeled F(ab')2 fragments of anti-Slr (small gold) and anti-M1 (large gold) IgG. The F(ab')2 fragments were incubated with wild type and mutant strains and analyzed by negative staining and transmission electron microscopy. The AP1 strain, expressing both Slr and M1 protein, bound both M1 and Slr antibodies simultaneously, (Figure 3C) whereas the mutant MB1 strain only showed binding of M1 antibodies verifying that the Slr protein is not expressed on the surface (Figure 3C). The mutant MC25 strain only exhibited binding of Slr antibodies, showing that the M1 protein is not present on the bacterial surface (Figure 3C). The expression pattern of Slr and M1 was investigated in the wild type strain AP1. The growth was plotted as OD620 versus the incubation time (Figure 4A), and samples were taken for Western blot analysis. Slr protein can be detected from hour 6 (approx. OD620 = 0.4) and declines at hour 9 (approx. OD620 = 0.8), while M1 can be detected from hour 2 (approx. OD620 = 0.05) (Figure 4B) and starts to be degraded at hour 7 (data not shown). Taken together, these results indicate that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with the M1 protein in early stationary growth phase.


The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen.

Bober M, Mörgelin M, Olin AI, von Pawel-Rammingen U, Collin M - PLoS ONE (2011)

Slr gene disruption, mutant strain analysis and protein localization by electron microscopy.A: Schematic representation of the mutagenesis strategy used to disrupt slr in S. pyogenes strain AP1. B: SDS-PAGE and Western blot analysis, using antibodies against Slr and M1, of bacterial cell extracts of the mutant strains MB1 and MC25, and wild type bacteria AP1. C: Wild type strain AP1, mutant strain MB1 and mutant strain MC25 incubated with gold labeled F(ab')2 fragments of anti-Slr (small gold particles) and anti-M1 (big gold particles) IgG. The dots represent binding of the F(ab')2 fragments to the Slr and M1 protein on the bacterial surface. Arrowheads without arrows indicate binding of anti-Slr antibodies, and complete arrows indicate binding of anti-M1 antibodies in all three panels. Scale bar  = 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105044&req=5

pone-0020345-g003: Slr gene disruption, mutant strain analysis and protein localization by electron microscopy.A: Schematic representation of the mutagenesis strategy used to disrupt slr in S. pyogenes strain AP1. B: SDS-PAGE and Western blot analysis, using antibodies against Slr and M1, of bacterial cell extracts of the mutant strains MB1 and MC25, and wild type bacteria AP1. C: Wild type strain AP1, mutant strain MB1 and mutant strain MC25 incubated with gold labeled F(ab')2 fragments of anti-Slr (small gold particles) and anti-M1 (big gold particles) IgG. The dots represent binding of the F(ab')2 fragments to the Slr and M1 protein on the bacterial surface. Arrowheads without arrows indicate binding of anti-Slr antibodies, and complete arrows indicate binding of anti-M1 antibodies in all three panels. Scale bar  = 100 nm.
Mentions: The Slr protein was expressed in E. coli using the GST Gene Fusion system and was used in several in vitro experiments (See Material and Methods). GST-Slr and Slr were separated on a SDS-PAGE for size and purity control. The GST-Slr has an apparent size of 110 kDa and Slr of approximately 85 kDa (Figure 2C). To confirm that Slr is a lipoprotein, a globomycin inhibition assay was performed. The maturation of lipoproteins is governed by signal peptidase II that removes signal peptides in the NH2-terminal signal sequence. This step is inhibited by globomycin, resulting in a surplus of signal peptides and thus an increased molecular size (Figure 2D), confirming that Slr is a lipoprotein. To investigate the role of Slr on the surface of S. pyogenes, we constructed a Slr mutant by single crossover mutagenesis of the AP1 strain of M1 serotype. A 505 bp promotorless fragment of the slr gene was cloned into multiple cloning site I of pFW13, resulting in vector pMB01 (Figure 3A). One mutant strain denoted MB1 was chosen for further analysis. Correct insertion of pMB01 was confirmed by PCR using gene- and plasmid specific primers (Figure 3A, primer 1–4, data not shown). To confirm slr disruption in MB1 and the Slr and M1 expression in AP1 and MC25, protein extracts from AP1, MB1 and MC25 cell pellets were analyzed by immunoblotting using anti-Slr and anti-M1 antibodies. A strong signal was detected in the AP1 pellet for both Slr and M1 protein. The only detectable signal in MB1 was for the M1 protein while in MC25 only Slr expression was detected (Figure 3B). Further mutant strain analysis and surface protein localization was done using gold labeled F(ab')2 fragments of anti-Slr (small gold) and anti-M1 (large gold) IgG. The F(ab')2 fragments were incubated with wild type and mutant strains and analyzed by negative staining and transmission electron microscopy. The AP1 strain, expressing both Slr and M1 protein, bound both M1 and Slr antibodies simultaneously, (Figure 3C) whereas the mutant MB1 strain only showed binding of M1 antibodies verifying that the Slr protein is not expressed on the surface (Figure 3C). The mutant MC25 strain only exhibited binding of Slr antibodies, showing that the M1 protein is not present on the bacterial surface (Figure 3C). The expression pattern of Slr and M1 was investigated in the wild type strain AP1. The growth was plotted as OD620 versus the incubation time (Figure 4A), and samples were taken for Western blot analysis. Slr protein can be detected from hour 6 (approx. OD620 = 0.4) and declines at hour 9 (approx. OD620 = 0.8), while M1 can be detected from hour 2 (approx. OD620 = 0.05) (Figure 4B) and starts to be degraded at hour 7 (data not shown). Taken together, these results indicate that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with the M1 protein in early stationary growth phase.

Bottom Line: Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance.In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface.This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection Medicine, Department of Clinical Sciences, Biomedical Center, Lund University, Lund, Sweden. marta.bober@med.lu.se

ABSTRACT
Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.

Show MeSH
Related in: MedlinePlus