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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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Binding of Slr to immobilized collagen I.A: Collagen I denatured with 4 M guanidine hydrochloride (+) and non-denatured collagen I (−) were applied to a PVDF membrane and incubated with radiolabeled Slr. The amount of collagen is indicated to the right. B: Binding of gold labeled Slr (the dots) and collagen I using electron microscopy. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the monomeric collagen I (panel I). The short span between light strands represents the overlap region, whiles the dark span represents the gap region on a collagen I fibril. Binding can be observed in both regions, but is mostly concentrated to the overlap region (panel II). Scale bar  = 100 nm.
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pone-0020345-g006: Binding of Slr to immobilized collagen I.A: Collagen I denatured with 4 M guanidine hydrochloride (+) and non-denatured collagen I (−) were applied to a PVDF membrane and incubated with radiolabeled Slr. The amount of collagen is indicated to the right. B: Binding of gold labeled Slr (the dots) and collagen I using electron microscopy. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the monomeric collagen I (panel I). The short span between light strands represents the overlap region, whiles the dark span represents the gap region on a collagen I fibril. Binding can be observed in both regions, but is mostly concentrated to the overlap region (panel II). Scale bar  = 100 nm.

Mentions: Previous results implicated that M proteins can interact with collagens (data not shown). Since it has been stipulated that LRRs have collagen binding capacity and since Slr is co-expressed with M protein on the bacterial surface there are reasons to investigate whether Slr can contribute to the collagen binding capacity of S. pyogenes. Therefore, we analyzed a possible Slr- collagen interaction. Wild type AP1 (expressing both M protein and Slr), mutant strain MB1 (lacking Slr protein), and MC25 (lacking M1 protein) bacteria were incubated with collagen I and the bound collagen was eluted with low pH glycine. The eluted proteins were immobilized on a PDVF membrane and anti-collagen Western blot was performed. The wild type AP1 strains exhibited strongest binding ability towards collagen I, while MB1 and MC25 strain showed diminished binding capacity (Figure 5A). The eluted collagen I from the AP1, MB1 and MC25 strains was also separated on 3–12% SDS-PAGE with collagen I as a standard and similar results could be observed as for anti-collagen Western blot, with AP1 showing a higher binding of collagen I than both MC25 and MB1 (Figure 5B). In order to further establish the collagen binding capacity of strains AP1, MB1 and MC25, a binding assay with radiolabeled collagen I was performed. The strains were incubated with 125I labeled collagen I, unbound collagen was removed by centrifugation and the radioactivity value of bound collagen was measured. All strains exhibited binding toward collagen I with the binding capacity of 36.8% for AP1, 36.1% for MB1 and 33.2% for MC25 (Figure 5C). The binding of collagen to the bacterial strains was also visualized by electron microscopy, where association of collagen I could be observed for all three strains with a tendency of less accumulation in MB1 and MC25 (Figure 5D). To determine the influence of collagen denaturation during SDS-PAGE on the Slr binding, non-denatured and guanidine hydrochloride-denatured collagen I was examined in a slot blot experiment. The same procedure was used to determine that the binding was not caused by the GST-tag on Slr. The slot blot experiment showed no interaction between collagen I and the GST-tag (data not shown). The radiolabeled Slr bound to both collagen preparations in a similar manner (Figure 6A). Further in vitro interaction between Slr and both monomer and fibrillar collagen I was investigated. Gold labeled Slr (the dots) and monomer collagen I show four binding sites. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the collagen (Figure 6B, panel I). More detailed analysis is required to identify the specific site of interaction. The collagen I fibril is composed of triple helices overlapping each other, resulting in a variation of triple helical thickness in the molecule. The short span between light strands represents the overlap region where the collagen triple helices overlap each other, while the dark span represents the gap region where the gap between collagen helices occurs. Binding can be observed in both regions, but is mostly concentrated to the overlap region (Figure 6B, panel II). These experiments were also executed with gold-labeled GST. There was no interactions between GST and monomeric or fibrillar collagen I (data not shown). The binding of Slr and M1 protein as well as the AP1, MB1 and MC25 strains to collagen I was investigated using surface plasmon resonance. Slr and M1 showed a dose-dependent binding to collagen I with a KD of 12 nM and 54 nM, respectively (Figure 7A and B). The bacterial strains AP1, MB1 and MC25 also showed a dose-dependent binding to collagen I (Figure 7C–E). Taken together, these results suggest that both Slr and M1 protein can bind collagen I, both when in purified form and in a bacterial surface context. Slr has a somewhat higher affinity towards collagen I than M1.


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)

Binding of Slr to immobilized collagen I.A: Collagen I denatured with 4 M guanidine hydrochloride (+) and non-denatured collagen I (−) were applied to a PVDF membrane and incubated with radiolabeled Slr. The amount of collagen is indicated to the right. B: Binding of gold labeled Slr (the dots) and collagen I using electron microscopy. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the monomeric collagen I (panel I). The short span between light strands represents the overlap region, whiles the dark span represents the gap region on a collagen I fibril. Binding can be observed in both regions, but is mostly concentrated to the overlap region (panel II). Scale bar  = 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105044&req=5

pone-0020345-g006: Binding of Slr to immobilized collagen I.A: Collagen I denatured with 4 M guanidine hydrochloride (+) and non-denatured collagen I (−) were applied to a PVDF membrane and incubated with radiolabeled Slr. The amount of collagen is indicated to the right. B: Binding of gold labeled Slr (the dots) and collagen I using electron microscopy. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the monomeric collagen I (panel I). The short span between light strands represents the overlap region, whiles the dark span represents the gap region on a collagen I fibril. Binding can be observed in both regions, but is mostly concentrated to the overlap region (panel II). Scale bar  = 100 nm.
Mentions: Previous results implicated that M proteins can interact with collagens (data not shown). Since it has been stipulated that LRRs have collagen binding capacity and since Slr is co-expressed with M protein on the bacterial surface there are reasons to investigate whether Slr can contribute to the collagen binding capacity of S. pyogenes. Therefore, we analyzed a possible Slr- collagen interaction. Wild type AP1 (expressing both M protein and Slr), mutant strain MB1 (lacking Slr protein), and MC25 (lacking M1 protein) bacteria were incubated with collagen I and the bound collagen was eluted with low pH glycine. The eluted proteins were immobilized on a PDVF membrane and anti-collagen Western blot was performed. The wild type AP1 strains exhibited strongest binding ability towards collagen I, while MB1 and MC25 strain showed diminished binding capacity (Figure 5A). The eluted collagen I from the AP1, MB1 and MC25 strains was also separated on 3–12% SDS-PAGE with collagen I as a standard and similar results could be observed as for anti-collagen Western blot, with AP1 showing a higher binding of collagen I than both MC25 and MB1 (Figure 5B). In order to further establish the collagen binding capacity of strains AP1, MB1 and MC25, a binding assay with radiolabeled collagen I was performed. The strains were incubated with 125I labeled collagen I, unbound collagen was removed by centrifugation and the radioactivity value of bound collagen was measured. All strains exhibited binding toward collagen I with the binding capacity of 36.8% for AP1, 36.1% for MB1 and 33.2% for MC25 (Figure 5C). The binding of collagen to the bacterial strains was also visualized by electron microscopy, where association of collagen I could be observed for all three strains with a tendency of less accumulation in MB1 and MC25 (Figure 5D). To determine the influence of collagen denaturation during SDS-PAGE on the Slr binding, non-denatured and guanidine hydrochloride-denatured collagen I was examined in a slot blot experiment. The same procedure was used to determine that the binding was not caused by the GST-tag on Slr. The slot blot experiment showed no interaction between collagen I and the GST-tag (data not shown). The radiolabeled Slr bound to both collagen preparations in a similar manner (Figure 6A). Further in vitro interaction between Slr and both monomer and fibrillar collagen I was investigated. Gold labeled Slr (the dots) and monomer collagen I show four binding sites. The dots are bound to C- and N terminal as well as 70 respectively 100 nm further in on the collagen (Figure 6B, panel I). More detailed analysis is required to identify the specific site of interaction. The collagen I fibril is composed of triple helices overlapping each other, resulting in a variation of triple helical thickness in the molecule. The short span between light strands represents the overlap region where the collagen triple helices overlap each other, while the dark span represents the gap region where the gap between collagen helices occurs. Binding can be observed in both regions, but is mostly concentrated to the overlap region (Figure 6B, panel II). These experiments were also executed with gold-labeled GST. There was no interactions between GST and monomeric or fibrillar collagen I (data not shown). The binding of Slr and M1 protein as well as the AP1, MB1 and MC25 strains to collagen I was investigated using surface plasmon resonance. Slr and M1 showed a dose-dependent binding to collagen I with a KD of 12 nM and 54 nM, respectively (Figure 7A and B). The bacterial strains AP1, MB1 and MC25 also showed a dose-dependent binding to collagen I (Figure 7C–E). Taken together, these results suggest that both Slr and M1 protein can bind collagen I, both when in purified form and in a bacterial surface context. Slr has a somewhat higher affinity towards collagen I than M1.

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