Strain-specific restriction of the antiphagocytic property of group A streptococcal M proteins

Abstract

Group A streptococcal M proteins are type-specific virulence factors that inhibit phagocytosis. We used two M proteins, M5 and Emm22, to analyze the influence of genetic background on the properties of M proteins. Mutant strains, engineered to lack these M proteins, were complemented with genes encoding the homologous or heterologous M protein, and the complemented strains were analyzed for phagocytosis resistance. Neither the M5 nor the Emm22 protein conferred phagocytosis resistance in the heterologous background, but they did do so in the homologous background. This was not due to lack of surface expression in the heterologous background. Moreover, the M5 and Emm22 proteins expressed in heterologous background appeared to have normal structure, since they were not affected in their ability to bind different human plasma proteins. In particular, M5 or Emm22 had normal ability to bind human complement inhibitors, a property that has been implicated in phagocytosis resistance. Results similar to those obtained with M5 and Emm22 were obtained in experiments with the M6 and Emm4 proteins. Together, these data suggest that the surface expression of M protein alone may not be sufficient to confer phagocytosis resistance and consequently that strain-specific factors other than M and Emm proteins may contribute to the ability of group A streptococci to resist phagocytosis.

FIG. 1

Schematic representation of the mga5 and mga22 regulons and the mutants used in this study. (A) Organization of the mga regulons of the type 5 strain M5 Manfredo and the type 22 strain AL168. Replacement of the emm5 gene with a gene encoding kanamycin resistance (Ωkm2) by homologous recombination gave rise to strain ΔM5 (10). In the AL168(mrp emm) derivative, the mrp22 gene has been disrupted by insertion of the conjugative transposon Tn916, whereas a large part of the emm22 gene has been replaced by a kanamycin casette (32). The mga gene encodes the multigene activator of group A streptococcus, and the mrp, emm, and enn genes encode proteins in the M protein family, whereas the scpA gene encodes the streptococcal C5a peptidase. The regions in the proteins marked A, B, and C are distinct domains containing three to five different repeat units. (B) Organization of the mga regulon of the ΔM5/emm22 derivative of the M5 Manfredo strain, in which the emm5 gene has been replaced by the emm22 gene. Protein ligands that are relevant for this study and their binding sites in M5 (Fg [fibrinogen] and FHL-1) and Emm22 (IgA and C4BP) are indicated.

FIG. 2

Binding of purified ligands to streptococci expressing the M5 and Emm22 proteins. The binding of ¹²⁵I-labeled ligands to the indicated group A streptococcal strains was measured as a function of bacterial number. Fibrinogen (A) and FHL-1 (B) are known to interact specifically with the M5 protein, whereas C4BP (C) and IgA (D) bind to the Emm22 protein. The data presented are based on three independent experiments with triplicate samples. The variation was <5% of the binding, and therefore error bars have not been introduced.

FIG. 3

Growth of group A streptococcal strains in human blood. The multiplication factor indicated on the ordinate represents the factor of the increase in number of CFU during a 3-h incubation in rotating tubes. (A) Experiments with strains expressing the M5 protein or no M protein (strain ΔM5). (B) Experiments with strains expressing the Emm22 protein or no M protein (AL168mrp⁻emm⁻). The data are also shown in Table 1.

FIG. 4

Binding of host ligands to an M-negative type 5 expressing Emm22 and OF22. The emm5 gene of M5 Manfredo was replaced by emm22, resulting in strain ΔM5/emm22. (A) This strain expresses Emm22 on its surface, as shown by its ability to bind C4BP and IgA. The gene encoding the opacity factor (sof) was cloned from the type M22 strain AL168 and inserted in pLZ12Spec. The resulting plasmid was then introduced into ΔM5/emm22, producing strain ΔM5/emm22(pLZsof22). (B) The ability of ΔM5/emm22(pLZsof22) to bind fibronectin, a ligand for OF, was compared with the fibronectin-binding capacity of the M5 Manfredo, ΔM5, and ΔM5/emm22 strains.