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Protein GRAB of streptococcus pyogenes regulates proteolysis at the bacterial surface by binding alpha2-macroglobulin
M Rasmussen1, H P Müller, L Björck
1Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, S-221 00 Lund, Sweden.
Abstract:
In the molecular interplay between pathogenic microorganisms and their host, proteolytic mechanisms are believed to play a crucial role. Here we find that the important human pathogen Streptococcus pyogenes (group A Streptococcus) expresses a surface protein with high affinity (Ka = 2.0 x 10(8) M-1) for alpha2-macroglobulin (alpha2M), the dominating proteinase inhibitor of human plasma. The immunoglobulin-binding protein G of group C and G streptococci also contains an alpha2M-binding domain and a gene encoding protein GRAB (protein G-related alpha2M-binding protein) was identified in the S. pyogenes Genome Sequencing data base. The grab gene is present in most S. pyogenes strains and is well conserved. Protein GRAB has typical features of a surface-attached protein of Gram-positive bacteria. It also contains a region homologous to parts of the alpha2M-binding domain of protein G and a variable number of a unique 28-amino acid-long repeat. Using Escherichia coli-produced protein GRAB and synthetic GRAB peptides, the alpha2M-binding region was mapped to the NH2-terminal part of protein GRAB, which is the region with homology to protein G. An isogenic S. pyogenes mutant lacking surface-associated protein GRAB showed no alpha2M binding activity and was attenuated in virulence when injected intraperitoneally in mice. Finally, alpha2M bound to the bacterial surface via protein GRAB was found to entrap and inhibit the activity of both S. pyogenes and host proteinases, thereby protecting important virulence determinants from proteolytic degradation. This regulation of proteolytic activity at the bacterial surface should affect the host-microbe relation during S. pyogenes infections.
Insights
Streptococcus pyogenes uses protein GRAB to bind alpha2-macroglobulin (alpha2M), a human proteinase inhibitor. This interaction protects bacterial virulence factors from degradation and reduces S. pyogenes virulence in mice.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- Proteolytic mechanisms are key in host-pathogen interactions.
- Streptococcus pyogenes (group A Streptococcus) is a significant human pathogen.
- Alpha2-macroglobulin (alpha2M) is the primary proteinase inhibitor in human plasma.
Purpose of the Study:
- To investigate the role of Streptococcus pyogenes surface proteins in interacting with host proteinase inhibitors.
- To identify and characterize a novel alpha2M-binding protein in S. pyogenes.
- To elucidate the function of this protein in bacterial virulence and host-pathogen dynamics.
Main Methods:
- Genome sequencing to identify the gene encoding the alpha2M-binding protein (GRAB).
- Biochemical assays using recombinant protein GRAB and synthetic peptides to map the alpha2M-binding site.
- Construction of an isogenic S. pyogenes mutant lacking GRAB.
- Virulence studies in a mouse model.
- Analysis of proteinase activity at the bacterial surface.
Main Results:
- Identified and characterized protein G-related alpha2M-binding protein (GRAB) in S. pyogenes, which binds alpha2M with high affinity.
- The grab gene is conserved among most S. pyogenes strains.
- The alpha2M-binding region of GRAB was mapped to its NH2-terminal part.
- A S. pyogenes mutant lacking GRAB exhibited no alpha2M binding and reduced virulence in mice.
- Surface-bound alpha2M inhibited both bacterial and host proteinases, protecting virulence factors.
Conclusions:
- Protein GRAB mediates the binding of alpha2M to the S. pyogenes surface.
- This interaction plays a significant role in S. pyogenes virulence by regulating proteolytic activity.
- GRAB-mediated alpha2M binding represents a novel mechanism for host-pathogen interaction and immune evasion.