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Streptococcus pyogenes (the group A streptococcus [GAS]) is an important human pathogen that commonly causes nasopharyngitis (strep throat), impetigo, and tonsillitis, as well as severe infections, such as necrotizing fasciitis, pyomyositis, bacterial sepsis, and streptococcal toxic shock syndrome. In addition, some GAS infections result in the nonsuppurative sequelae of rheumatic fever and acute glomerulonephritis. Although GAS is almost exclusively a human pathogen, a serotype M50 strain was isolated from infections in mice (7). This strain, B514, has recently been used in mouse models to investigate virulence factors of the GAS that contribute to disease (8, 9).

Mga is a multiple-gene regulator of GAS which activates several virulence genes, including those encoding the M protein (emm) and its relatives, the complement C5a peptidase (scpA), and the secreted inhibitor of complement (sic) (3, 10, 16, 19, 22, 23). Mga is also required for transcription of its own gene (mga) (15). The Mga sequence shows some similarity to those of response regulators of two-component sensor-transducer systems that are required for the expression of many genes in bacteria. Like response regulators, Mga is environmentally regulated and it has been shown to bind DNA upstream of several of the genes which it activates (11, 12). However, Mga is about twice as large as most proteins of this type and has three potential helix-turn-helix (HTH) motifs in its N terminus and two potential response regulator motifs at its C-terminal end (16, 18).

The mga gene exists in two major types that diverge from each other by approximately 22%. One type, characterized by mga1, is generally associated with mga regulons containing emm genes of subfamily 1, which includes emm1, emm6, emm5, and emm24, among others. The second type, characterized by mga4, is generally associated with mga regulons containing emm genes of subfamily 2, which includes emm2, emm4, emm49, and emm5, among others. These two forms of mga regulons appear to reflect a differing evolutionary history for the virulence loci and, in most instances, are concordant with the preferred tissue site for colonization in a host (2, 5, 6). Despite the considerable divergence between the major mga types, it has been shown that the two types have overlapping functions by the functional complementation of a deleted mga1 allele with a cloned mga4 allele (1).

In the M50 mouse-virulent GAS strain, there is very little transcription of either mrp (M-related protein) or emm and, consequently, there is a low level of M protein in this strain (24). Because the strain is M deficient, it is not surprising that a deletion of the emm gene had no consequences for virulence in the mouse models for long-term throat colonization or for pneumonia (9). We suggested that a defect in the mga gene in this strain might be the cause of this low transcription level. To test this idea, we have sequenced the mga gene from the M50 strain and determined that there are amino acid differences from other Mga proteins that could make it dysfunctional. We then used wild-type mga alleles from two different GAS strains to complement the mga allele resident in the M50 strain.

Sequencing and analysis of the mga gene from B514. The complete nucleotide sequence of the structural gene for mga from strain B514, called mga50, was determined from PCR-generated DNA fragments made with B514 chromosomal DNA as the template. The open reading frame encodes 533 amino acids. As expected, mga50 differs substantially (22% divergence) from the mga1 gene of the JRS4 M6 serotype strain (mga regulon type 1) (16). It is closely related to mga genes that are derived from the second form of mga (2% divergence from mga4 and mga49). To decipher potential deficiencies in this particular mga50 allele, it was compared to the homologous mga4 and mga49 alleles from strains AP4 and CS101, respectively (Fig. 1) (1, 18).

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Comparison of amino acid sequences from different GAS mga gene sequences. Mga50 was aligned via the Clustal W algorithm with Mga4 (1) and Mga49 (VirR49) (18). Positions of identical residues are marked with a dash. Polymorphic residues are noted, with bold type indicating those residues at which Mga50 varies from both Mga4 and Mga49. Two potential HTH motifs at the amino terminus are marked as boxes. Two possible domains for sensor proteins to recognize are located at the carboxy terminus, and they are at residues 172 to 300 and residue 404 to the end of the protein, as described by Perez-Casal et al. (16). Residues 484 to 499 differ due to a frameshift in mga49 relative to the sequences of the other two alleles.

Analysis of transcripts following complementation. To establish whether a functional defect in mga50 is indeed responsible for the attenuated expression of the mga-regulated genes, a complementation test was performed. The mga gene (mga4) from a serotype 4 strain, AP4, was cloned into the shuttle vector pLZ12-Spec (1) and electroporated into the M50 strain B514. Quantitative RNA slot blotting was performed to measure the amounts of the transcripts of emm50 and mga50. Methods were previously described (13, 24). RNA was isolated from different strains during exponential growth. RNA in 2- and 0.4-µg amounts was loaded onto filters in duplicate. Blots were reacted with emm- or mga-specific DNA and then stripped for hybridization with the recA probe. Hybridized counts were detected by a phosphorimager. The amounts of RNA in the blots were normalized with a recA transcript and compared to those in a standard strain, T2/44/RB4, which was previously found to make normal levels of M protein (24).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Transcript levels in B514 compared to those in T2/44/RB4 and complemented strains. Transcripts specific for mga, emm, and recA were detected in strains T2/44/RB4 (T2), B514, B514/pMga4 (pMga4), and B514/pLZ12-Spec (pLZ12). Normalized units are defined as the band intensity measured for mga or emm probe at a particular RNA concentration divided by the band intensity of a recA internal control probe at the same RNA concentration (13).

Restoration of wild-type levels of M family proteins in strain B514 by complementation with mga-containing plasmids. It had been suggested that the decreased transcription of the emm gene results in a lower level of M protein on the surface of B514 (24). This was tested by comparing the amount of M protein in CNBr extracts of strain B514 with strains complemented with different mga alleles expressed under different promoters (Fig. 3). Complementation by the mga4 allele under its own promoter has been described (1). The divergent mga1 gene from a serotype M6 (mga1 from strain D471) GAS strain was cloned into the shuttle vector pLZ12-Spec and expressed either under its own promoter or under one derived from the Lactococcus lactis phage SKIIG (1, 17). As previously described (24), M50 was barely detectable in B514 and in B514/pLZ12-Spec (Fig. 3, lanes 2 and 3). However, both the mga4 and mga1 genes complement the mga50 allele for the production of M50. For mga1, complementation occurs both from the native mga1 promoter (Fig. 3, lanes 4 and 5) and from a constitutive phage promoter (13) (lane 6). CNBr extracts were made by the method of Raeder et al. (21) from in vitro-grown cultures with an absorbance of 0.6 at 600 nm. The number of cells per extract was normalized, and the bands in each extract were visualized on sodium dodecyl sulfate gels by Coomassie blue staining. The detection of measurable amounts of M proteins in complemented strains shows that the heterologous Mga proteins are functional and the amount of M50 protein on the cell surface increases when the amount of emm transcript increases.

View larger version (105K): [in this window] [in a new window]   FIG. 3.   CNBr extracts of surface proteins from B514, T2/44/RB4, and plasmid-transformed B514 cells. Stds, standards. B514/pLZ12, B514/pLZ12-Spec.

Antiphagocytic function of M50 protein. The antiphagocytic function of the M protein of GAS is considered to be a critical virulence factor because it enables the bacteria to resist killing by polymorphonuclear leukocytes. The hyaluronic capsule of GAS also performs an antiphagocytic function in some strains (4, 14). Complementation of Mga increased the levels of M and M-related proteins in B514 from the low basal level to substantial levels approximately equivalent to those in fresh human clinical isolates. To demonstrate that complementation also restores resistance to opsonophagocytosis, we used a direct bactericidal assay in whole human blood. For this, we used a derivative of B514, B514.039, in which the hasA gene required to form a capsule had been insertionally inactivated (9). This allowed the test of function to be completed in the absence of a capsule, a second potential mediator of antiphagocytic resistance.

Nucleotide sequence accession number. The sequence of mga50 was deposited in GenBank under accession no. AF071802.