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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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Visualization and confirmation of the LRR lipoprotein Slr.A: Slr protein visualized by electron microscopy showing the typical horseshoe shape of a LRR protein. Scale bar  = 25 nm. B: a homology model of Slr using InlA as the template. The 11 LRRs forming β-sheets in yellow, α−helices in blue, and loops in turquoise. C: SDS-PAGE analysis of recombinantly expressed GST-Slr and Slr with the GST tag cleaved off. D: Western blot analysis, using anti-Slr antibodies, of bacterial cell extracts from AP1 bacteria grown in the presence of increasing amounts of the signal peptidase II inhibitor globomycin as indicated.
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pone-0020345-g002: Visualization and confirmation of the LRR lipoprotein Slr.A: Slr protein visualized by electron microscopy showing the typical horseshoe shape of a LRR protein. Scale bar  = 25 nm. B: a homology model of Slr using InlA as the template. The 11 LRRs forming β-sheets in yellow, α−helices in blue, and loops in turquoise. C: SDS-PAGE analysis of recombinantly expressed GST-Slr and Slr with the GST tag cleaved off. D: Western blot analysis, using anti-Slr antibodies, of bacterial cell extracts from AP1 bacteria grown in the presence of increasing amounts of the signal peptidase II inhibitor globomycin as indicated.

Mentions: The slr gene encoding the S. pyogenes LRR protein Slr is present in 32/32 strains of 24 different M serotypes as determined by PCR (Table 1) and is also present in all currently sequenced S. pyogenes genomes. The slr gene in strain AP1 was cloned and sequenced using oligonucleotide primers based on the genome sequence from the M1 strain SF370 (Accession no. NC_002737) [37]. Sequencing of this gene revealed an ORF encoding a 793 amino acid protein that is 99% identical to the predicted Slr protein from SF370. The sequences of the Slr from AP1 and the corresponding gene slr, have been deposited in Genbank under the accession no. HQ908654. Slr contains a 21 amino acid putative N-terminal signal sequence ending with the amino acid sequence TLIA. This is the recognition sequence for signal peptidase II that allows for lipid modification of the resulting amino-terminal cysteine and insertion into the cellular membrane. In between amino acid 22 and 421 are four histidine triad motifs, that are not present in InlA of L. monocytogenes. There are 13 LRRs in Slr spanning over amino acid numbers 421–705 forming the β-sheets compared to 15 LRRs in InlA. The carboxyl terminal end of Slr contains histidine rich repeat sequences, but lacks a cell wall anchoring motif, while InlA carries a classical LPtTG cell wall anchoring motif (Figure 1A). The LRR in Slr resembles the consensus motif of the LRR region in InlA (Figure 1B). To confirm the horseshoe shape of the protein, Slr was visualized by electron microscopy. The Slr is pointed out by arrows and the shape is well visible supporting a horseshoe like conformation of Slr (Figure 2A). Homology modeling of Slr using InlA as the template further supported the protein's structure. The modeled LRRs forming the β-sheets (blue) are suggested to be the protein-protein interaction sites (Figure 2B).


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)

Visualization and confirmation of the LRR lipoprotein Slr.A: Slr protein visualized by electron microscopy showing the typical horseshoe shape of a LRR protein. Scale bar  = 25 nm. B: a homology model of Slr using InlA as the template. The 11 LRRs forming β-sheets in yellow, α−helices in blue, and loops in turquoise. C: SDS-PAGE analysis of recombinantly expressed GST-Slr and Slr with the GST tag cleaved off. D: Western blot analysis, using anti-Slr antibodies, of bacterial cell extracts from AP1 bacteria grown in the presence of increasing amounts of the signal peptidase II inhibitor globomycin as indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105044&req=5

pone-0020345-g002: Visualization and confirmation of the LRR lipoprotein Slr.A: Slr protein visualized by electron microscopy showing the typical horseshoe shape of a LRR protein. Scale bar  = 25 nm. B: a homology model of Slr using InlA as the template. The 11 LRRs forming β-sheets in yellow, α−helices in blue, and loops in turquoise. C: SDS-PAGE analysis of recombinantly expressed GST-Slr and Slr with the GST tag cleaved off. D: Western blot analysis, using anti-Slr antibodies, of bacterial cell extracts from AP1 bacteria grown in the presence of increasing amounts of the signal peptidase II inhibitor globomycin as indicated.
Mentions: The slr gene encoding the S. pyogenes LRR protein Slr is present in 32/32 strains of 24 different M serotypes as determined by PCR (Table 1) and is also present in all currently sequenced S. pyogenes genomes. The slr gene in strain AP1 was cloned and sequenced using oligonucleotide primers based on the genome sequence from the M1 strain SF370 (Accession no. NC_002737) [37]. Sequencing of this gene revealed an ORF encoding a 793 amino acid protein that is 99% identical to the predicted Slr protein from SF370. The sequences of the Slr from AP1 and the corresponding gene slr, have been deposited in Genbank under the accession no. HQ908654. Slr contains a 21 amino acid putative N-terminal signal sequence ending with the amino acid sequence TLIA. This is the recognition sequence for signal peptidase II that allows for lipid modification of the resulting amino-terminal cysteine and insertion into the cellular membrane. In between amino acid 22 and 421 are four histidine triad motifs, that are not present in InlA of L. monocytogenes. There are 13 LRRs in Slr spanning over amino acid numbers 421–705 forming the β-sheets compared to 15 LRRs in InlA. The carboxyl terminal end of Slr contains histidine rich repeat sequences, but lacks a cell wall anchoring motif, while InlA carries a classical LPtTG cell wall anchoring motif (Figure 1A). The LRR in Slr resembles the consensus motif of the LRR region in InlA (Figure 1B). To confirm the horseshoe shape of the protein, Slr was visualized by electron microscopy. The Slr is pointed out by arrows and the shape is well visible supporting a horseshoe like conformation of Slr (Figure 2A). Homology modeling of Slr using InlA as the template further supported the protein's structure. The modeled LRRs forming the β-sheets (blue) are suggested to be the protein-protein interaction sites (Figure 2B).

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