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Distinct Single Amino Acid Replacements in the Control of Virulence Regulator Protein Differentially Impact Streptococcal Pathogenesis

DOI: 10.1371/journal.ppat.1002311

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Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 ? resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis.


[1]  Toledo-Arana A, Dussurget O, Nikitas G, Sesto N, Guet-Revillet H, et al. (2009) The Listeria transcriptional landscape from saprophytism to virulence. Nature 459: 950–956.
[2]  Talaat AM, Lyons R, Howard ST, Johnston SA (2004) The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc Natl Acad Sci U S A 101: 4602–4607.
[3]  Wright JS 3rd, Jin R, Novick RP (2005) Transient interference with staphylococcal quorum sensing blocks abscess formation. Proc Natl Acad Sci U S A 102: 1691–1696.
[4]  Kazmierczak MJ, Wiedmann M, Boor KJ (2005) Alternative sigma factors and their roles in bacterial virulence. Microbiol Mol Biol Rev 69: 527–543.
[5]  Beier D, Gross R (2006) Regulation of bacterial virulence by two-component systems. Curr Opin Microbiol 9: 143–152.
[6]  Ramirez-Pena E, Trevino J, Liu Z, Perez N, Sumby P (2010) The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript. Mol Microbiol 78: 1332–1347.
[7]  Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69: 183–215.
[8]  Rasko DA, Moreira CG, Li de R, Reading NC, Ritchie JM, et al. (2008) Targeting QseC signaling and virulence for antibiotic development. Science 321: 1078–1080.
[9]  Malhotra V, Sharma D, Ramanathan VD, Shakila H, Saini DK, et al. (2004) Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett 231: 237–245.
[10]  Sitkiewicz I, Musser JM (2006) Expression microarray and mouse virulence analysis of four conserved two-component gene regulatory systems in group A Streptococcus. Infect Immun 74: 1339–1351.
[11]  Musser JM, Shelburne SA 3rd (2009) A decade of molecular pathogenomic analysis of group A Streptococcus. J Clin Invest 119: 2455–2463.
[12]  Olsen RJ, Shelburne SA, Musser JM (2009) Molecular mechanisms underlying group A streptococcal pathogenesis. Cell Microbiol 11: 1–12.
[13]  Kreikemeyer B, McIver KS, Podbielski A (2003) Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions. Trends Microbiol 11: 224–232.
[14]  Churchward G (2007) The two faces of Janus: virulence gene regulation by CovR/S in group A streptococci. Mol Microbiol 64: 34–41.
[15]  Kenney LJ (2002) Structure/function relationships in OmpR and other winged-helix transcription factors. Curr Opin Microbiol 5: 135–141.
[16]  Churchward G, Bates C, Gusa AA, Stringer V, Scott JR (2009) Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site. Microbiology 155: 566–575.
[17]  Gao J, Gusa AA, Scott JR, Churchward G (2005) Binding of the global response regulator protein CovR to the sag promoter of Streptococcus pyogenes reveals a new mode of CovR-DNA interaction. J Biol Chem 280: 38948–38956.
[18]  Levin JC, Wessels MR (1998) Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus. Mol Microbiol 30: 209–219.
[19]  Engleberg NC, Heath A, Miller A, Rivera C, DiRita VJ (2001) Spontaneous mutations in the CsrRS two-component regulatory system of Streptococcus pyogenes result in enhanced virulence in a murine model of skin and soft tissue infection. J Infect Dis 183: 1043–1054.
[20]  Miyoshi-Akiyama T, Ikebe T, Watanabe H, Uchiyama T, Kirikae T, et al. (2006) Use of DNA arrays to identify a mutation in the negative regulator, csrR, responsible for the high virulence of a naturally occurring type M3 group A Streptococcus clinical isolate. J Infect Dis 193: 1677–1684.
[21]  Beres SB, Carroll RK, Shea PR, Sitkiewicz I, Martinez-Gutierrez JC, et al. (2010) Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics. Proc Natl Acad Sci U S A 107: 4371–4376.
[22]  Trevino J, Perez N, Ramirez-Pena E, Liu Z, Shelburne SA 3rd, et al. (2009) CovS simultaneously activates and inhibits the CovR-mediated repression of distinct subsets of group A Streptococcus virulence factor-encoding genes. Infect Immun 77: 3141–3149.
[23]  Shea PR, Beres SB, Flores AR, Ewbank AL, Gonzalez-Lugo JH, et al. (2011) Distinct signatures of diversifying selection revealed by genome analysis of respiratory tract and invasive bacterial populations. Proc Natl Acad Sci U S A 108: 5039–5044.
[24]  Ikebe T, Ato M, Matsumura T, Hasegawa H, Sata T, et al. (2010) Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates. PLoS Pathog 6: e1000832.
[25]  Nowak E, Panjikar S, Konarev P, Svergun DI, Tucker PA (2006) The structural basis of signal transduction for the response regulator PrrA from Mycobacterium tuberculosis. J Biol Chem 281: 9659–9666.
[26]  Buckler DR, Zhou Y, Stock AM (2002) Evidence of intradomain and interdomain flexibility in an OmpR/PhoB homolog from Thermotoga maritima. Structure 10: 153–164.
[27]  Hendrickson WA, Horton JR, LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. Embo J 9: 1665–1672.
[28]  Holm L, Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38: W545–549.
[29]  Blanco AG, Sola M, Gomis-Ruth FX, Coll M (2002) Tandem DNA recognition by PhoB, a two-component signal transduction transcriptional activator. Structure 10: 701–713.
[30]  Carroll RK, Shelburne SA 3rd, Olsen RJ, Suber B, Sahasrabhojane P, et al. (2011) Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice. J Clin Invest 121: 1956–1968.
[31]  Federle MJ, Scott JR (2002) Identification of binding sites for the group A streptococcal global regulator CovR. Mol Microbiol 43: 1161–1172.
[32]  Gryllos I, Grifantini R, Colaprico A, Jiang S, Deforce E, et al. (2007) Mg(2+) signalling defines the group A streptococcal CsrRS (CovRS) regulon. Mol Microbiol 65: 671–683.
[33]  Manetti AG, Zingaretti C, Falugi F, Capo S, Bombaci M, et al. (2007) Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation. Mol Microbiol 64: 968–983.
[34]  Sun H, Ringdahl U, Homeister JW, Fay WP, Engleberg NC, et al. (2004) Plasminogen is a critical host pathogenicity factor for group A streptococcal infection. Science 305: 1283–1286.
[35]  Nizet V, Beall B, Bast DJ, Datta V, Kilburn L, et al. (2000) Genetic locus for streptolysin S production by group A Streptococcus. Infect Immun 68: 4245–4254.
[36]  Graham MR, Smoot LM, Migliaccio CA, Virtaneva K, Sturdevant DE, et al. (2002) Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc Natl Acad Sci U S A 99: 13855–13860.
[37]  Edwards RJ, Taylor GW, Ferguson M, Murray S, Rendell N, et al. (2005) Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes. J Infect Dis 192: 783–790.
[38]  Podbielski A, Woischnik M, Leonard BA, Schmidt KH (1999) Characterization of nra, a global negative regulator gene in group A streptococci. Mol Microbiol 31: 1051–1064.
[39]  Nakagawa I, Kurokawa K, Yamashita A, Nakata M, Tomiyasu Y, et al. (2003) Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution. Genome Res 13: 1042–1055.
[40]  Beres SB, Sylva GL, Barbian KD, Lei B, Hoff JS, et al. (2002) Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc Natl Acad Sci U S A 99: 10078–10083.
[41]  Sumby P, Porcella SF, Madrigal AG, Barbian KD, Virtaneva K, et al. (2005) Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events. J Infect Dis 192: 771–782.
[42]  Turner CE, Kurupati P, Jones MD, Edwards RJ, Sriskandan S (2009) Emerging role of the interleukin-8 cleaving enzyme SpyCEP in clinical Streptococcus pyogenes infection. J Infect Dis 200: 555–563.
[43]  Chen SL, Hung CS, Pinkner JS, Walker JN, Cusumano CK, et al. (2009) Positive selection identifies an in vivo role for FimH during urinary tract infection in addition to mannose binding. Proc Natl Acad Sci U S A 106: 22439–22444.
[44]  Rhee JE, Sheng W, Morgan LK, Nolet R, Liao X, et al. (2008) Amino acids important for DNA recognition by the response regulator OmpR. J Biol Chem 283: 8664–8677.
[45]  Doi A, Okajima T, Gotoh Y, Tanizawa K, Utsumi R (2010) X-ray crystal structure of the DNA-binding domain of response regulator WalR essential to the cell viability of Staphylococcus aureus and interaction with target DNA. Biosci Biotechnol Biochem 74: 1901–1907.
[46]  Maris AE, Walthers D, Mattison K, Byers N, Kenney LJ (2005) The response regulator OmpR oligomerizes via beta-sheets to form head-to-head dimers. J Mol Biol 350: 843–856.
[47]  Roberts SA, Churchward GG, Scott JR (2007) Unraveling the regulatory network in Streptococcus pyogenes: the global response regulator CovR represses rivR directly. J Bacteriol 189: 1459–1463.
[48]  Graham MR, Virtaneva K, Porcella SF, Barry WT, Gowen BB, et al. (2005) Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies. Am J Pathol 166: 455–465.
[49]  Graham MR, Virtaneva K, Porcella SF, Gardner DJ, Long RD, et al. (2006) Analysis of the transcriptome of group A Streptococcus in mouse soft tissue infection. Am J Pathol 169: 927–942.
[50]  Shelburne SA, Olsen RJ, Suber B, Sahasrabhojane P, Sumby P, et al. (2010) A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection. PLoS Pathog 6: e1000817.
[51]  Kratovac Z, Manoharan A, Luo F, Lizano S, Bessen DE (2007) Population genetics and linkage analysis of loci within the FCT region of Streptococcus pyogenes. J Bacteriol 189: 1299–1310.
[52]  Crotty Alexander LE, Maisey HC, Timmer AM, Rooijakkers SH, Gallo RL, et al. (2010) M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment. J Mol Med 88: 371–381.
[53]  Kreth J, Chen Z, Ferretti J, Malke H (2011) Counteractive balancing of transcriptome expression involving CodY and CovRS in Streptococcus pyogenes. J Bacteriol 193: 4153–4165.
[54]  McShan WM, Ferretti JJ, Karasawa T, Suvorov AN, Lin S, et al. (2008) Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes. J Bacteriol 190: 7773–7785.
[55]  Federle MJ, McIver KS, Scott JR (1999) A response regulator that represses transcription of several virulence operons in the group A Streptococcus. J Bacteriol 181: 3649–3657.
[56]  Leslie AG (2006) The integration of macromolecular diffraction data. Acta Crystallogr D Biol Crystallogr 62: 48–57.
[57]  Terwilliger TC, Berendzen J (1999) Discrimination of solvent from protein regions in native Fouriers as a means of evaluating heavy-atom solutions in the MIR and MAD methods. Acta Crystallogr D Biol Crystallogr 55: 501–505.
[58]  Terwilliger T (1999) Reciprocal-space solvent flattening. Acta Crystallogr D Biol Crystallogr 55: 1863–1871.
[59]  Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.
[60]  Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, et al. (1998) Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination. Acta Crystallogr D Biol Crystallogr 54: 905–921.
[61]  DeLano WL (2002) The PyMol Molecular Graphics System. DeLano Scientific, Palo Alto, CA, USA.
[62]  Shelburne SA 3rd, Keith D, Horstmann N, Sumby P, Davenport MT, et al. (2008) A direct link between carbohydrate utilization and virulence in the major human pathogen group A Streptococcus. Proc Natl Acad Sci U S A 105: 1698–1703.
[63]  Virtaneva K, Porcella SF, Graham MR, Ireland RM, Johnson CA, et al. (2005) Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques. Proc Natl Acad Sci U S A 102: 9014–9019.
[64]  Heath A, DiRita VJ, Barg NL, Engleberg NC (1999) A two-component regulatory system, CsrR-CsrS, represses expression of three Streptococcus pyogenes virulence factors, hyaluronic acid capsule, streptolysin S, and pyrogenic exotoxin B. Infect Immun 67: 5298–5305.
[65]  Hoe NP, Vuopio-Varkila J, Vaara M, Grigsby D, De Lorenzo D, et al. (2001) Distribution of streptococcal inhibitor of complement variants in pharyngitis and invasive isolates in an epidemic of serotype M1 group A Streptococcus infection. J Infect Dis 183: 633–639.
[66]  Gryllos I, Tran-Winkler HJ, Cheng MF, Chung H, Bolcome R 3rd, et al. (2008) Induction of group A Streptococcus virulence by a human antimicrobial peptide. Proc Natl Acad Sci U S A 105: 16755–16760.
[67]  Smoot JC, Barbian KD, Van Gompel JJ, Smoot LM, Chaussee MS, et al. (2002) Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc Natl Acad Sci U S A 99: 4668–4673.
[68]  Beres SB, Musser JM (2007) Contribution of exogenous genetic elements to the group A Streptococcus metagenome. PLoS ONE 2: e800.
[69]  Li J, Kasper DL, Ausubel FM, Rosner B, Michel JL (1997) Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus. Proc Natl Acad Sci U S A 94: 13251–13256.


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