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Functional dissection of Streptococcus pyogenes M5 protein: the hypervariable region is essential for virulence.

Waldemarsson J, Stålhammar-Carlemalm M, Sandin C, Castellino FJ, Lindahl G - PLoS ONE (2009)

Bottom Line: Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection.The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg.In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Lund, Sweden.

ABSTRACT
The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

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Schematic representation of the M5 protein and its ligand binding properties.(A) The M5 protein can be divided into three distinct regions: the N-terminal hypervariable region (HVR), the B-repeat region and the C-repeat region [19]. Each of these regions binds a human plasma protein, as indicated. In-frame deletions in the chromosomal emm5 gene results in expression of the ΔN1, ΔN2, ΔB, and ΔC deletion variants. After removal of the signal peptide, M5 has a total length of 450 aa residues. Part of the C-repeat region and the C-terminal region are probably hidden in the bacterial cell wall [2]. The numbers indicate aa residues in the processed form of M5. FHL-1, factor H-like protein 1; Fg, fibrinogen. Adapted from refs. [22] and [25]. (B) Ligand binding properties of bacterial mutants used in this study. The ΔN1 and ΔN2 mutants do not bind FHL-1, the ΔB mutant does not bind Fg and the ΔC mutant is unable to bind albumin, while the regions not deleted in these mutants retain their ligand-binding properties. Based on data in ref. [25].
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pone-0007279-g001: Schematic representation of the M5 protein and its ligand binding properties.(A) The M5 protein can be divided into three distinct regions: the N-terminal hypervariable region (HVR), the B-repeat region and the C-repeat region [19]. Each of these regions binds a human plasma protein, as indicated. In-frame deletions in the chromosomal emm5 gene results in expression of the ΔN1, ΔN2, ΔB, and ΔC deletion variants. After removal of the signal peptide, M5 has a total length of 450 aa residues. Part of the C-repeat region and the C-terminal region are probably hidden in the bacterial cell wall [2]. The numbers indicate aa residues in the processed form of M5. FHL-1, factor H-like protein 1; Fg, fibrinogen. Adapted from refs. [22] and [25]. (B) Ligand binding properties of bacterial mutants used in this study. The ΔN1 and ΔN2 mutants do not bind FHL-1, the ΔB mutant does not bind Fg and the ΔC mutant is unable to bind albumin, while the regions not deleted in these mutants retain their ligand-binding properties. Based on data in ref. [25].

Mentions: The M5 protein comprises three well-defined regions: the HVR, the B-repeats and the C-repeats (Figure 1A). Previous work has shown that each of these regions binds a human plasma protein. The HVR binds FHL-1, a splice variant of the complement regulator factor H (FH) [23]. The role of bound FHL-1 remains unknown [24], [25], and mice do not have FHL-1 (P. Zipfel, personal communication), implying that any role of the HVR in S. pyogenes M5 infected mice cannot be explained through binding of FHL-1. The B-repeat region binds Fg and is required for phagocytosis resistance, making it the only region in M5 with a well-defined function, as determined ex vivo [21], [22], [25]. The C-repeat region binds albumin [25]–[27], and also promotes binding to CD46, a surface-localized complement regulator present on all human cells [8], [11]. It is not known whether M5 and other M proteins bind CD46 of mouse origin, but even if this were the case, the limited tissue distribution of CD46 in normal mice [28] makes it unlikely that the interaction would be of relevance in the mouse model.


Functional dissection of Streptococcus pyogenes M5 protein: the hypervariable region is essential for virulence.

Waldemarsson J, Stålhammar-Carlemalm M, Sandin C, Castellino FJ, Lindahl G - PLoS ONE (2009)

Schematic representation of the M5 protein and its ligand binding properties.(A) The M5 protein can be divided into three distinct regions: the N-terminal hypervariable region (HVR), the B-repeat region and the C-repeat region [19]. Each of these regions binds a human plasma protein, as indicated. In-frame deletions in the chromosomal emm5 gene results in expression of the ΔN1, ΔN2, ΔB, and ΔC deletion variants. After removal of the signal peptide, M5 has a total length of 450 aa residues. Part of the C-repeat region and the C-terminal region are probably hidden in the bacterial cell wall [2]. The numbers indicate aa residues in the processed form of M5. FHL-1, factor H-like protein 1; Fg, fibrinogen. Adapted from refs. [22] and [25]. (B) Ligand binding properties of bacterial mutants used in this study. The ΔN1 and ΔN2 mutants do not bind FHL-1, the ΔB mutant does not bind Fg and the ΔC mutant is unable to bind albumin, while the regions not deleted in these mutants retain their ligand-binding properties. Based on data in ref. [25].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749438&req=5

pone-0007279-g001: Schematic representation of the M5 protein and its ligand binding properties.(A) The M5 protein can be divided into three distinct regions: the N-terminal hypervariable region (HVR), the B-repeat region and the C-repeat region [19]. Each of these regions binds a human plasma protein, as indicated. In-frame deletions in the chromosomal emm5 gene results in expression of the ΔN1, ΔN2, ΔB, and ΔC deletion variants. After removal of the signal peptide, M5 has a total length of 450 aa residues. Part of the C-repeat region and the C-terminal region are probably hidden in the bacterial cell wall [2]. The numbers indicate aa residues in the processed form of M5. FHL-1, factor H-like protein 1; Fg, fibrinogen. Adapted from refs. [22] and [25]. (B) Ligand binding properties of bacterial mutants used in this study. The ΔN1 and ΔN2 mutants do not bind FHL-1, the ΔB mutant does not bind Fg and the ΔC mutant is unable to bind albumin, while the regions not deleted in these mutants retain their ligand-binding properties. Based on data in ref. [25].
Mentions: The M5 protein comprises three well-defined regions: the HVR, the B-repeats and the C-repeats (Figure 1A). Previous work has shown that each of these regions binds a human plasma protein. The HVR binds FHL-1, a splice variant of the complement regulator factor H (FH) [23]. The role of bound FHL-1 remains unknown [24], [25], and mice do not have FHL-1 (P. Zipfel, personal communication), implying that any role of the HVR in S. pyogenes M5 infected mice cannot be explained through binding of FHL-1. The B-repeat region binds Fg and is required for phagocytosis resistance, making it the only region in M5 with a well-defined function, as determined ex vivo [21], [22], [25]. The C-repeat region binds albumin [25]–[27], and also promotes binding to CD46, a surface-localized complement regulator present on all human cells [8], [11]. It is not known whether M5 and other M proteins bind CD46 of mouse origin, but even if this were the case, the limited tissue distribution of CD46 in normal mice [28] makes it unlikely that the interaction would be of relevance in the mouse model.

Bottom Line: Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection.The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg.In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Lund, Sweden.

ABSTRACT
The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

Show MeSH
Related in: MedlinePlus