Identification of Residues Responsible for the Defective Virulence Gene Regulator Mga Produced by a Natural Mutant ofStreptococcus pyogenes

Abstract

Mga is a transcriptional regulator in the pathogen Streptococcus pyogenes that positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of "gain-of-function" mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen.

Domains Required for Transcriptional Activation Show Conservation in the Mga Family of Virulence Gene Regulators

“…The S. pyogenes vectors for integration (VIT) strain RTG229 is a derivative of JRS4 (7). Both the M6 Pemm-gusA GAS reporter strain KSM148.174 and the M6 Pmrp-gusA GAS reporter strain KSM149 are unmarked mga deletion (⌬mga) derivatives of the VIT strain RTG229 (27). The clinical isolate AP4 is a serotype M4 GAS strain (25).…”

“…Mutagenic PCR was performed across the 5Ј region of mga using the GeneMorph PCR mutagenesis kit (Stratagene) on plasmid pKSM162 (16) with primers RMut1-R2 and Mga6-150 (Table 1) and an initial amount of DNA of 0.110 g. The resulting PCR product was digested with AvaI and SpeI, ligated with AvaI-and SpeI-digested pKSM318 (27), transformed into E. coli DH5␣, and grown overnight in 10 ml LB broth containing spectinomycin. A similar procedure was performed for the 3Ј portion of mga except primers OYR-29 and RMut2-L were used on various concentrations of pKSM162 ranging from 0.0552 to 0.838 g. The reduction in starting concentration was to compensate for the increased size of the 3Ј fragment as per the manufacturer's protocol.…”

“…Pmga-gusA reporter strain KSM231.310 was generated by transforming the mgadeleted M6 VIT strain VIT231 with pPmga-gusA (27) linearized by XmnI, which created a ⌬mga reporter strain containing a single-copy transcriptional fusion of the native M6 mga promoter to gusA (Pmga-gusA).…”

“…Plasmid DNA was extracted from the entire overnight culture, and 1 g of plasmid DNA was transformed into the reporter strain KSM148.174 (27). Following selection on THY agar containing spectinomycin and 175 g/ml 5-bromo-4-chloro-3-indolyl-␤-D-glucuronic acid (X-Gluc) (Gold Biotechnology), GAS transformants that were either light blue or white on plates were subjected to Western analysis to determine protein levels as described below.…”

“…Two amino-terminal helix-turnhelix (HTH) domains necessary for DNA binding and transcriptional activation were found in M6 Mga: one (HTH-4) that is absolutely essential for binding to all targets and a second (HTH-3) that serves an accessory role, primarily in autoregulation from Pmga (16). In addition, two amino acids involved in transcriptional activation of a divergent Mga (mga-2) have been identified (27). Finally, two putative response regulator receiver domains have been suggested to exist in Mga based on homology to other two-component systems (20); however, no molecular evidence for these domains has been presented.…”