Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence

Abstract

Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

FIG 1

mga and emm are expressed at maximal levels during exponential phases of growth. Transcript levels of mga and emm in strain MGAS15249 were measured by qRT-PCR. Samples were collected at the indicated growth phases for transcript analysis. Three biological replicates were grown and analyzed in triplicate. Data graphed are means ± standard deviations. Values with single asterisks (P < 0.01) and double asterisks (P < 0.001) indicate statistically significantly different transcript levels compared to the late exponential phase of growth.

FIG 2

Genome-wide transcript analysis of the Δmga strain compared to the wild type. (A) Gene expression in the MGAS15249 Δmga strain. The relative gene expression level in the Δmga:pDC strain compared to the Δmga:pDC-mga strain is shown. Mean fold changes in transcript levels of differentially regulated genes and their respective genomic coordinates are shown (P < 0.05 compared to the wild type, determined by Baggerly's test after applying Bonferroni's correction for multiple comparisons). (B to D) Genetic arrangement of significantly regulated genes in the Δmga:pDC strain (genes with a >10-fold change in transcript levels).

FIG 3

Modeling studies of Mga. (A) Schematic representation of the domain architecture of Mga. Individual domains are labeled, and the domain boundaries are indicated by the amino acid numbering at the bottom. The locations of phosphorylatable histidines are indicated and labeled. (B) Ribbon representation of the Mga model, as predicted by I-TASSER, superimposed on the structure of AtxA from B. anthracis (PDB accession number 4R6I). Individual domains of Mga are color-coded as shown in panel A. Individual domains of AtxA are color-coded as follows: the N-terminal domain (NTD) is in yellow, PRD-1 is in brown, PRD-2 is in light blue, and the EIIB domain is in light pink. An additional C-terminal fragment in Mga is indicated in green. Phosphorylatable histidines, H207 and H273 of PRD-1 and H327 of PRD-2, are shown as spheres and marked by arrows. A phosphorylatable histidine (H199) in PRD-1 of AtxA is shown as an orange sphere. (C) Ribbon representation of the model of the Mga dimer. One subunit is color-coded as described above for panel B, and the second subunit is shown in gray. The orientation of the model in panel C is tilted and rotated on the x axis relative to that in panel B for a better representation of the dimer.

FIG 4

Mga is phosphorylated in vivo. The isogenic mga mutant strain trans-complemented with an empty vector (Δmga:pDC), the wild-type mga strain (Δmga:pDC-mga), or the hexahistidine-tagged wild-type mga strain (Δmga:pDC-mga-His₆) was grown in THY medium to the late exponential growth phase (A₆₀₀ of ∼1.0). Cells were lysed, and cell lysates were clarified by methanol precipitation. (A) Equal concentrations of cell lysates of the Δmga:pDC (lane 1), Δmga:pDC-mga (lane 2), and Δmga:pDC-mga-His₆ strains were resolved on a 10% SDS-PAGE gel and probed by monoclonal antihexahistidine antibodies. (B) Equal concentrations of cell lysates of the Δmga:pDC strain (lane 1), the Δmga:pDC-mga-His₆ strain (lane 2), and the Δmga:pDC-mga-His₆ strain treated with hydroxylamine (lane 3) were resolved on a 10% SDS-PAGE gel containing 50 μM Phos-tag and ZnSO₄ and probed by monoclonal antihexahistidine antibodies. Bands corresponding to nonphosphorylated Mga (Mga-His₆) and phosphorylated Mga (Mga-P-His₆) are labeled and indicated by arrows.

FIG 5

Phosphorylation- and non-phosphorylation-mimicking substitutions at H201 of PRD-1 of Mga do not alter transcript levels of Mga-regulated genes. Transcript levels of mga, emm, scpA, and sclA were measured by qRT-PCR. Four biological replicates were grown and analyzed in triplicate. Data graphed are means ± standard deviations.

FIG 6

Phosphorylation- and non-phosphorylation-mimicking substitutions at H207 and H273 of PRD-1 of Mga alter transcript levels of Mga-regulated genes. Isogenic mutant strains with single (A) or double (B) phosphorylation- and non-phosphorylation-mimicking substitutions at H207 and H273 of PRD-1 were grown to the exponential phase of growth. Transcript levels of mga, emm, scpA, and sclA in the indicated strains were measured by qRT-PCR. Four biological replicates were grown and analyzed in triplicate. Data graphed are means ± standard deviations. Values with single asterisks (P < 0.05) and double asterisks (P < 0.0005) indicate statistically significantly different transcript levels compared to the wild type.

FIG 7

Phosphorylation- and non-phosphorylation-mimicking substitutions at H327 of PRD-2 of Mga alter transcript levels of Mga-regulated genes. Transcript levels of mga, emm, scpA, and sclA in the indicated strains were measured by qRT-PCR. Four biological replicates were grown and analyzed in triplicate. Data graphed are means ± standard deviations. Values with single asterisks (P < 0.05) and double asterisks (P < 0.0005) indicate statistically significantly different transcript levels compared to the wild type.

FIG 8

Phosphorylation/non-phosphorylation-mimicking substitutions at H207 and H273 of PRD-1 of Mga alter the global mga transcriptional profile. (A) Heat map depicting the alterations in the gene expression pattern at the global level in the indicated strains. (B) Heat map of transcript levels of selected genes in the indicated strains relative to the wild type. The scheme for the color values is shown at the bottom. Red in the key represents downregulated genes, black represents genes not detected/not differentially expressed, and green represents upregulated genes in the indicated strains relative to the wild type.

FIG 9

In vivo phosphorylation status of the wild type and various alanine mutants of Mga, as assessed by Phos-tag immunoblot analysis. Equal concentrations of cell lysates from the indicated strains were resolved on a 10% SDS-PAGE gel containing 50 μM Phos-tag and ZnSO₄ and probed by monoclonal antihexahistidine antibodies. Bands corresponding to nonphosphorylated Mga (Mga-His₆) and phosphorylated Mga (Mga-P-His₆) are labeled and indicated by arrows.

FIG 10

Phosphorylation- and non-phosphorylation-mimicking substitutions at H273 of PRD-1 of Mga alter M59 GAS virulence. (A) Twenty mice were infected with each indicated strain intramuscularly, and near-mortality was graphed as a Kaplan-Meier survival curve. Statistical significance between strains, as assessed by a log rank test, is indicated. (B) Histological examination of muscular lesions from mice infected with the indicated strains. The confined, less destructive lesions are indicated by arrowheads, and the areas of damaged muscle tissue with more destructive lesions are boxed. (C) Twenty mice were infected intramuscularly, and mean CFU recovered from the infected muscle tissue are shown, with P values being determined by a t test.

FIG 11

Phosphorylation- and non-phosphorylation-mimicking substitutions at H207 of PRD-1 of Mga attenuate M59 GAS virulence. Twenty mice were infected with each indicated strain intramuscularly, and near-mortality was graphed as a Kaplan-Meier survival curve. Statistical significance between strains, as assessed by a log rank test, is indicated.