Restoration of Mga Function to a<i>Streptococcus pyogenes</i>Strain (M Type 50) That Is Virulent in Mice

Limbago, Brandi; McIver, Kevin S.; Penumalli, Vikram; Weinrick, Brian; Scott, June R.

doi:10.1128/iai.69.2.1215-1220.2001

Cited by 9 publications

(8 citation statements)

References 60 publications

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“…Based upon the location of a naturally occurring asparagine (N26) to serine (S26) substitution in the the HTH‐1 motif of the defective serotype M50 Mga, this domain was thought to be important for Mga DNA‐binding activity (Yung et al. , 1999; Limbago et al , 2001). However, the mutational analysis described here indicates that the HTH‐1 motif (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The HTH‐3 (pKSM118.3) and HTH‐4 (pKSM118.4) mutations were transferred into pJRS2050 through exchange of a 364 bp Sna BI– Spe I restriction fragment to produce pKSM120.3 and pKSM120.4 respectively. pJRS9160 (Limbago et al , 2001) is a derivative of the erythromycin‐resistant gene replacement vector pJRS233 containing the wild‐type GAS rpsL gene from serotype M6 D471. The 4.5 kb Bam HI– Hin dIII fragment containing either HTH‐3 (pKSM120.3) or HTH‐4 (pKSM120.4) along with flanking homologous sequences were cloned into pJRS9160 to produce pKSM168.3 and pKSM168.4 respectively.…”

Section: Methodsmentioning

confidence: 99%

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Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus

McIver¹,

Myles

2002

Molecular Microbiology

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Summary Mga is a DNA‐binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix–turn–helix DNA‐binding motifs (HTH1–HTH4) identified within the amino‐terminus of Mga, alanine substitutions were introduced within each domain in a MBP–Mga fusion allele and purified proteins were assayed for binding to Mga‐specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH‐1 and HTH‐2 mutations showed wild type DNA‐binding activity, an altered HTH‐3 domain resulted in reduced binding to the three promoters and an HTH‐4 mutant was devoid of detectable binding activity. Plasmid‐encoded expression of the HTH‐3 and HTH‐4 alleles from a constitutive promoter (Pspac) in the mga‐deleted GAS strain JRS519 demonstrated that Mga‐regulated emm expression correlated directly to the DNA‐binding activity observed for each mutant protein in vitro. Single‐copy expression of HTH‐3 and HTH‐4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA‐binding domains (HTH‐3 and HTH‐4) that are required for direct activation of the Mga virulence regulon in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus

McIver¹,

Myles

2002

Molecular Microbiology

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“…The S. pyogenes vectors for integration (VIT) strain RTG229 is a derivative of the serotype M6 strain JRS4 (10,27). B514-Sm is a spontaneous streptomycin-resistant derivative of the serotype M50 GAS strain B514 (17). The clinical isolate AP4 is a serotype M4 GAS strain (28).…”

Section: Methodsmentioning

confidence: 99%

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

Vahling

McIver

2005

J Bacteriol

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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.

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“…Present among all GAS strains are mga alleles belonging to either one of two main lineages exhibiting ~ 28% divergence, whereby the classical throat strains have one lineage form (mga-1) and classical skin strains harbor the other form (mga-2) [12]. The role of Mga in virulence has been examined in numerous animal models for colonization and/or infection of the upper respiratory tract, and for invasive disease involving deeper soft tissue [11,[13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Role of Mga in group A streptococcal infection at the skin epithelium

Luo

Lizano

Banik

et al. 2008

Microbial Pathogenesis

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Group A streptococci (GAS) primarily cause infection at epithelial tissue sites of its human host. The role of the transcriptional regulator Mga in a humanized mouse model for superficial skin infection was investigated. Inactivation of mga in a skin strain (Alab49) led to loss of virulence. The Deltamga mutant displayed >100-fold decrease in emm (pam) transcript levels, and loss of bacterial-bound plasmin activity. A slight decrease in speB transcription, accompanied by a partial decrease in cysteine protease activity but no change in PrtF2 degradation, was also observed. Mga had no effect on transcription of nra, Nra-regulated pilus genes (cpa, fctA) or other FCT-region genes (msmR, prtF2). Combined with findings on other Alab49 mutants, data show that several essential virulence genes are regulated by Mga or Nra, but not both, implying that any coordinated response during skin infection likely operates at a higher level of transcriptional control. Mga was required for bacterial autoaggregation and biofilm-like growth on an abiotic surface; however, aggregation and biofilm formation have only partial overlap with the skin virulence phenotype. Findings on numerous phenotypes for 7 mutants constructed on the same genetic background yield a detailed, integrated model for GAS pathogenesis at the skin.

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Restoration of Mga Function to aStreptococcus pyogenesStrain (M Type 50) That Is Virulent in Mice

Cited by 9 publications

References 60 publications

Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus

Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus

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

Role of Mga in group A streptococcal infection at the skin epithelium

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