Structural and functional analysis of RopB: a major virulence regulator in S treptococcus pyogenes

Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB‐dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C‐terminal domain of RopB. RopB‐CTD has the TPR motif, a signature motif involved in protein–peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density‐specific cell‐free growth medium demonstrated the presence of a low molecular weight proteinaceous secreted factor that upregulates RopB‐dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure‐guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB‐dependent speB expression and attenuated GAS virulence. Results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials.

[1]  M. Federle,et al.  Rgg protein structure–function and inhibition by cyclic peptide compounds , 2015, Proceedings of the National Academy of Sciences.

[2]  R. Olsen,et al.  Adhesin competence repressor (AdcR) from Streptococcus pyogenes controls adaptive responses to zinc limitation and contributes to virulence , 2014, Nucleic acids research.

[3]  S. W. Long,et al.  Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences , 2014, Proceedings of the National Academy of Sciences.

[4]  A. Friedler,et al.  Allosteric modulation of protein oligomerization: an emerging approach to drug design , 2014, Front. Chem..

[5]  Yang Zhang,et al.  Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment , 2013, Bioinform..

[6]  S. Zouhir,et al.  Peptide-binding dependent conformational changes regulate the transcriptional activity of the quorum-sensor NprR , 2013, Nucleic acids research.

[7]  R. Grenha,et al.  Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR , 2012, Proceedings of the National Academy of Sciences.

[8]  Jiri Stulik,et al.  Tetratricopeptide Repeat Motifs in the World of Bacterial Pathogens: Role in Virulence Mechanisms , 2012, Infection and Immunity.

[9]  R. Olsen,et al.  Polymorphisms in regulator of protease B (RopB) alter disease phenotype and strain virulence of serotype M3 Group A Streptococcus , 2012, The Journal of infectious diseases.

[10]  R. Olsen,et al.  An amino‐terminal signal peptide of Vfr protein negatively influences RopB‐dependent SpeB expression and attenuates virulence in Streptococcus pyogenes , 2011, Molecular microbiology.

[11]  J. Musser,et al.  From transcription to activation: how group A streptococcus, the flesh‐eating pathogen, regulates SpeB cysteine protease production , 2011, Molecular microbiology.

[12]  R. Olsen,et al.  Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice. , 2011, The Journal of clinical investigation.

[13]  R. Gardan,et al.  Rgg proteins associated with internalized small hydrophobic peptides: a new quorum‐sensing mechanism in streptococci , 2011, Molecular microbiology.

[14]  G. Guarneros,et al.  The RNPP family of quorum-sensing proteins in Gram-positive bacteria , 2010, Applied Microbiology and Biotechnology.

[15]  D. Hoffmeister,et al.  Spotlights on advances in mycotoxin research , 2010, Applied Microbiology and Biotechnology.

[16]  Yuriy Fofanov,et al.  Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics , 2010, Proceedings of the National Academy of Sciences.

[17]  Vincent B. Chen,et al.  PHENIX: a comprehensive Python-based system for macromolecular structure solution , 2010, Acta crystallographica. Section D, Biological crystallography.

[18]  R. Olsen,et al.  Molecular pathogenesis of necrotizing fasciitis. , 2010, Annual review of pathology.

[19]  Adeline R. Whitney,et al.  Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis , 2010, Proceedings of the National Academy of Sciences.

[20]  A. Norrby-Teglund,et al.  Cathelicidin LL-37 in Severe Streptococcus pyogenes Soft Tissue Infections in Humans , 2008, Infection and Immunity.

[21]  S. Arold,et al.  Structure of PlcR: Insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria , 2007, Proceedings of the National Academy of Sciences.

[22]  M. Caparon,et al.  Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression , 2006, Journal of bacteriology.

[23]  D. Ohlendorf,et al.  Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Y. Lacasse,et al.  From the authors , 2005, European Respiratory Journal.

[25]  M. Neely,et al.  Role of RopB in Growth Phase Expression of the SpeB Cysteine Protease of Streptococcus pyogenes , 2003, Journal of bacteriology.

[26]  D. Lereclus,et al.  A cell–cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group , 2002, The EMBO journal.

[27]  G. Dunny,et al.  ccfA, the Genetic Determinant for the cCF10 Peptide Pheromone in Enterococcus faecalis OG1RF , 2002, Journal of bacteriology.

[28]  M. Cunningham,et al.  Pathogenesis of group A streptococcal infections. , 2000, Clinical microbiology reviews.

[29]  Thomas C. Terwilliger,et al.  Reciprocal-space solvent flattening , 1999, Acta crystallographica. Section D, Biological crystallography.

[30]  T. Terwilliger,et al.  Discrimination of solvent from protein regions in native Fouriers as a means of evaluating heavy-atom solutions in the MIR and MAD methods , 1999, Acta crystallographica. Section D, Biological crystallography.

[31]  J. Musser,et al.  Expression and Characterization of Group AStreptococcus Extracellular Cysteine Protease Recombinant Mutant Proteins and Documentation of Seroconversion during Human Invasive Disease Episodes , 1998, Infection and Immunity.

[32]  F. Ausubel,et al.  Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus , 1997 .

[33]  M. Chaussee,et al.  Temporal production of streptococcal erythrogenic toxin B (streptococcal cysteine proteinase) in response to nutrient depletion , 1997, Infection and immunity.

[34]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[35]  J. Musser,et al.  Vaccination with streptococcal extracellular cysteine protease (interleukin-1 beta convertase) protects mice against challenge with heterologous group A streptococci. , 1994, Microbial pathogenesis.

[36]  W. Hendrickson.  Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. , 1991, Science.

[37]  I. Olafsson,et al.  Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor , 1989, Nature.

[38]  A. Ralph,et al.  Group a streptococcal diseases and their global burden. , 2013, Current topics in microbiology and immunology.

[39]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[40]  S. Doublié.  Preparation of selenomethionyl proteins for phase determination. , 1997, Methods in enzymology.

[41]  S. Doublié.  [29] Preparation of selenomethionyl proteins for phase determination. , 1997, Methods in enzymology.

[42]  A G Leslie,et al.  Biological Crystallography Integration of Macromolecular Diffraction Data , 2022 .