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Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus

  • Paul E. Bernard
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas

    Texas A&M Health Science Center College of Medicine, Bryan, Texas
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  • Priyanka Kachroo
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • Jesus M. Eraso
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • Luchang Zhu
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • Jessica E. Madry
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • Sarah E. Linson
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • Matthew Ojeda Saavedra
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • Concepcion Cantu
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas
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  • James M. Musser
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas

    Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York
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  • Randall J. Olsen
    Correspondence
    Address correspondence to Randall J. Olsen, M.D., Ph.D., Houston Methodist Research Institute and Houston Methodist Hospital, 6565 Fannin St., MS: B490, Houston, TX 77030.
    Affiliations
    Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas

    Texas A&M Health Science Center College of Medicine, Bryan, Texas

    Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York
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Open ArchivePublished:July 29, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.06.009
      Two-component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine-tune their activity. In group A Streptococcus (GAS), regulator of Cov (RocA) is an accessory protein that functions with the control of virulence regulator/sensor TCS, which regulates approximately 15% of the GAS transcriptome. Whole-genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid–altering) and nonsense (protein-truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally occurring clinical isolates with rocA polymorphisms (n = 48), an isogenic rocA deletion mutant strain, and five isogenic rocA polymorphism mutant strains to perform genome-wide transcript analysis (RNA sequencing), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change, causing a wild-type–like virulence phenotype, or a substantial transcriptome change, leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, our data show that naturally occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.
      Two-component systems (TCSs) are signal transduction proteins that enable bacteria to rapidly adapt to external stimuli by altering gene expression.
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      Although its molecular mechanism of action is unknown, RocA increases the activity of CovR by increasing CovR phosphorylation in a CovS-dependent manner.
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      Regulatory rewiring confers serotype-specific hyper-virulence in the human pathogen group A Streptococcus.
      Putative functional domains of RocA have been mapped to the amino-terminal transmembrane domains,
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      suggesting membrane interactions of RocA and CovS are important for signal modulation (Figure 1A).
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      Figure thumbnail gr1
      Figure 1Clinical isolates with naturally occurring rocA polymorphisms have altered global transcriptomes. A: rocA is unusually polymorphic in serotype M28 GAS strains. The affected codon and amino acid change conferred by each polymorphism are shown. For polymorphisms due to nucleotide deletion, the affected nucleotide is identified. Alleles identified in multiple isolates are indicated. Polymorphisms that result in RocA protein truncation or presumed loss of rocA mRNA translation
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      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      are shown below the protein schematic, and polymorphisms that result in amino acid changes are shown above the protein schematic. Missense polymorphisms in the amino-terminal transmembrane domains are red. Predicted domains of the RocA protein are indicated.
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      Predicted functional domains of the putative histidine kinase domain (H box, N box, F box, and G box) are identified.
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      Identification of rocA, a positive regulator of covR expression in the group A streptococcus.
      Asterisks denote stop codons. Daggers indicate one strain has two polymorphisms in rocA. Adapted from Bernard et al.
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      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      Copyright © American Society for Microbiology. B and C: Three-dimensional principal component (PC) analysis of the RNA-sequencing data generated with the clinical isolates with naturally occurring rocA polymorphisms, an isogenic rocA deletion mutant (ΔrocA) strain, and four phylogenetically matched wild-type (WT) strains at midexponential (B) and early-stationary (C) growth phases. Clusters were determined by average-linkage hierarchical clustering and keyed by manual inspection of the data to determine common and differentiating features of strains within each cluster. D: Protein schematic highlighting RocA variants chosen for further study by generation of isogenic mutant strains. Asterisks denote stop codons. HATPase, histidine kinase ATPase domain; TM, transmembrane domain.
      Our interest in RocA is based on serotype M28 GAS whole-genome sequencing studies of strains collected from human invasive infections.
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      • Ramalinga A.B.
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      Regulatory rewiring confers serotype-specific hyper-virulence in the human pathogen group A Streptococcus.
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      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Jain I.
      • Miller E.W.
      • Danger J.L.
      • Pflughoeft K.J.
      • Sumby P.
      RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus.
      • Sarkar P.
      • Danger J.L.
      • Jain I.
      • Meadows L.A.
      • Beam C.
      • Medicielo J.
      • Burgess C.
      • Musser J.M.
      • Sumby P.
      Phenotypic variation in the group A Streptococcus due to natural mutation in the accessory protein-encoding gene rocA.
      we discovered that the number of missense (amino acid–altering) and nonsense (protein-truncating) polymorphisms in rocA within the M28 population studied was higher than expected (Figure 1A).
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      We hypothesized that naturally occurring polymorphisms in rocA result in altered RocA function and contribute to the molecular pathogenesis of serotype M28 GAS invasive infections. To test this hypothesis, clinical isolates and isogenic mutant strains were used to perform genome-wide transcript analysis [RNA sequencing (RNA-seq)], in vitro virulence factor assays, and mouse and nonhuman primate (NHP) pathogenesis studies.

      Materials and Methods

      RNA-Seq Analysis

      Clinical Isolates Grown in Duplicate

      GAS strains were grown in duplicate at 37°C with 5% CO2 in Todd-Hewitt broth with 0.2% yeast extract (THY) to midexponential (ME; OD600 = 0.5) and early-stationary (ES; OD600 = 1.65) growth phases, as previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Sanson M.
      • O'Neill B.E.
      • Kachroo P.
      • Anderson J.R.
      • Flores A.R.
      • Valson C.
      • Cantu C.C.
      • Makthal N.
      • Karmonik C.
      • Fittipaldi N.
      • Kumaraswami M.
      • Musser J.M.
      • Olsen R.J.
      A naturally occurring single amino acid replacement in multiple gene regulator of group A streptococcus significantly increases virulence.
      • Sanson M.
      • Makthal N.
      • Gavagan M.
      • Cantu C.
      • Olsen R.J.
      • Musser J.M.
      • Kumaraswami M.
      Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.
      RNAprotect Bacteria Reagent (Qiagen Inc., Germantown, MD) was added at a 2:1 ratio, and then cells were lysed by ballistic disintegration (FastPrep-96 instrument and lysing matrix B; MP Biomedicals, Santa Ana, CA). RNA was extracted using standard methods (RNeasy kit; Qiagen Inc.), and then RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit (Invitrogen, Carlsbad, CA), respectively. cDNA libraries were prepared using standard procedures [Ribo-Zero rRNA Removal Kit Bacteria (Illumina, San Diego, CA) and ScriptSeq Complete Kit (Epicentre, Madison, WI)]. The cDNA libraries were sequenced with an Illumina NextSeq500 instrument using the default settings.

      Isogenic rocA Mutant Strains Grown in Triplicate

      GAS strains were grown in triplicate at 37°C with 5% CO2 in THY to ME and ES growth phases, as previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Sanson M.
      • O'Neill B.E.
      • Kachroo P.
      • Anderson J.R.
      • Flores A.R.
      • Valson C.
      • Cantu C.C.
      • Makthal N.
      • Karmonik C.
      • Fittipaldi N.
      • Kumaraswami M.
      • Musser J.M.
      • Olsen R.J.
      A naturally occurring single amino acid replacement in multiple gene regulator of group A streptococcus significantly increases virulence.
      • Sanson M.
      • Makthal N.
      • Gavagan M.
      • Cantu C.
      • Olsen R.J.
      • Musser J.M.
      • Kumaraswami M.
      Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.
      RNAprotect Bacteria Reagent was added at a 2:1 ratio, and then cells were lysed by ballistic disintegration (FastPrep-96 instrument and lysing matrix B). RNA was extracted using standard methods (RNeasy kit), and then RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer and Qubit, respectively. Ribosomal RNA was removed with the MicrobExpress Kit (Ambion, Carlsbad, CA). cDNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA), according to the manufacturer's instructions. The cDNA libraries were then sequenced using an Illumina NextSeq500 instrument using the default settings.

      RNA-Seq Data Analysis

      On average, 14.9 million reads/sample were obtained for the phylogenetically matched wild-type (WT) strains, the isogenic rocA deletion mutant (ΔrocA) strain, and the clinical isolates with naturally occurring rocA polymorphisms (clinical isolate RNA-seq); and 42.6 million reads/sample were obtained for the parental WT strain and isogenic rocA mutant strains (isogenic rocA mutant RNA-seq). Reads were mapped to the serotype M28 GAS reference strain MGAS6180 genome
      • Green N.M.
      • Zhang S.
      • Porcella S.F.
      • Nagiec M.J.
      • Barbian K.D.
      • Beres S.B.
      • LeFebvre R.B.
      • Musser J.M.
      Genome sequence of a serotype M28 strain of group A Streptococcus: potential new insights into puerperal sepsis and bacterial disease specificity.
      with EDGE-pro,
      • Magoc T.
      • Wood D.
      • Salzberg S.L.
      EDGE-pro: estimated degree of gene expression in prokaryotic genomes.
      and then differential expression analysis was performed using DESeq2 version 1.14.1.
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      For the clinical isolate RNA-seq data, genes encoding rRNA, tRNA, phage, and mobile genetic elements were excluded from the analysis, as these elements are not present in every clinical isolate.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      For the isogenic rocA mutant RNA-seq data, genes encoding rRNA and tRNA were excluded from the analysis, along with genes from phage 6180.2 because the parental WT strain lacks this phage.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      Genes with an absolute transcript change of ≥1.5-fold and P < 0.05 after Wald test with Bonferroni correction for multiple comparisons were considered as significantly differentially expressed. For principal component analysis, clusters were determined by average-linkage hierarchical clustering and keyed by manual inspection of the data to determine common and differentiating features of strains within each cluster.

      Generation of Isogenic rocA Polymorphism Strains

      Strain MGAS28426 was selected as the serotype M28 parental WT strain because it is genetically representative of serotype M28 GAS strains and because it has a WT allele for the major global transcription regulators.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      In addition, this strain is also used in animal infection studies.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      The isogenic rocA deletion mutant strain has been previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      The isogenic rocA polymorphism mutant strains were constructed by allelic exchange (the allele of interest was cloned from a clinical isolate with the naturally occurring rocA polymorphism), as previously described.
      • Zhu L.
      • Olsen R.J.
      • Nasser W.
      • Beres S.B.
      • Vuopio J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      Primer sequences are listed in Table 1. Whole genome sequence analysis of the isogenic rocA mutant strains confirmed the expected rocA polymorphism and absence of spurious mutations.
      Table 1Primers Used in This Study
      PrimerSequenceRemarks
      rocA-3F5′-GCGCTAGCGAATTTCATTGGCGAATTGAC-3′Sanger sequencing flanking primer
      rocA-5F5′-GCGCGGTTTACCAAGAAGCTCAAGAGTAT-3′
      rocA-H60Y-15′-GCGTGGATCCGACGATCAAGTCGCTCTAACGGCTT-3′
      rocA-H60Y-25′-GCGTGGATCCTTCCTCAGGTTAAAACGGTTGCCT-3′
      rocA-H60Y-seq5′-AACCAAACTAATAGACACTAGTGGAAAAAAGGC-3′Sanger sequencing H60Y mutation
      rocA-VNTR-seq5′-CTGTTAGAATGACAGAACTTATGATA-5′Sanger sequencing -9del(6nt) mutation
      • Zhu L.
      • Olsen R.J.
      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      rocA-P97L-15′-GCGTGGATCCACAATTTGCCAGCTATTGGCGGGCA-3′
      rocA-P97L-25′-GCGTGGATCCAGACTATGGATTTGCGTTATCACCG-3′
      rocA-P97L-seq5′-GCTCTATGTGATTGAAAATATTGGCGCCAGG-3′Sanger sequencing P97L mutation
      rocA-G184W-15′-GCGTGGATCCGATGCCACCTTTTGGAAGAATCCAA-3′
      rocA-G184W-25′-GCGTGGATCCAGGCAGGGAAGGCAGAGGATATTAT-3′
      rocA-G184W-seq5′-GCCTCAATAGAGTTTTGTTTTACATAACGC-3′Sanger sequencing G184W mutation
      rocA-R258K-15′-GCGTGGATCCTGACATCACAGAACGCCAAGCAAGA-3′
      rocA-R258K-25′-GCGTGGATCCTGTAGACATGTTCCCCCATACAGCT-3′
      rocA-R258K-seq5′-CTAAACAAGTTAAATCAAGTCTGTCATCTTTAGC-3′Sanger sequencing R258K mutation
      rocA-T442P-15′-GCGTGGATCCGGACTCTCCCAATCTTTCCAAGTCA-3′
      rocA-T442P-25′-GCGTGGATCCAATATAGAAAAGTTACTTAATCAAG-3′
      rocA-T442P-seq5′-CCAACTTGGCAAAGCTGAAATTTTAACTCTAGC-3′Sanger sequencing T442P mutation
      tufA-TaqF5′-GACACGCGGACTACGTTAAA-3′tufA TaqMan primers and probes
      tufA-TaqR5′-CACCAACCTGACGTGAAAGA-3′
      tufA-TaqP5′-6FAM-TGACGATGTCAGTTGTCTTTGTTTGGA-TAMRA-3′
      fasX-TaqF5′-GATATGATGGCTCGGCAGAC-3′fasX TaqMan primers and probes
      fasX-TaqR5′-GCCGGGCTTTGATACTGAT-3′
      fasX-TaqP5′-6FAM-TGACTCACCTATCGAACAGCCTCGA-TAMRA-3′
      BamHI sites are in bold.
      Seq, sequencing; VNTR, variable number tandem repeat.

      Growth in Acidic Conditions

      GAS strains were grown using THY supplemented with 0.1 mol/L 2-(N-morpholino)ethanesulfonic acid (pH 6.0; Sigma Aldrich, St. Louis, MO), as previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.

      Western Immunoblot Analysis of SPN and SLO in Culture Supernatant

      Western immunoblot analysis of NAD+-glycohydrolase (SPN) and streptolysin O (SLO) was performed, as previously described, at ME growth phase.
      • Zhu L.
      • Olsen R.J.
      • Nasser W.
      • Beres S.B.
      • Vuopio J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      Total protein, as determined by a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA), was used as the loading control.

      Activity Assays

      SPN and SLO activity was measured, as previously described, using ME growth phase culture supernatants.
      • Zhu L.
      • Olsen R.J.
      • Lee J.D.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Contribution of secreted NADase and streptolysin O to the pathogenesis of epidemic serotype M1 Streptococcus pyogenes infections.
      Serum opacity factor (SOF) activity in overnight culture supernatants was assayed, as previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      Secreted streptococcal pyrogenic exotoxin B (SpeB) protease activity was assayed using the casein hydrolysis (milk plate) assay, as previously described.
      • Olsen R.J.
      • Raghuram A.
      • Cantu C.
      • Hartman M.H.
      • Jimenez F.E.
      • Lee S.
      • Ngo A.
      • Rice K.A.
      • Saddington D.
      • Spillman H.
      • Valson C.
      • Flores A.R.
      • Beres S.B.
      • Long S.W.
      • Nasser W.
      • Musser J.M.
      The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.
      Streptokinase (SKA) activity was measured, as previously described, using ME and ES growth phase cell-free culture supernatants.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Ramirez-Pena E.
      • Trevino J.
      • Liu Z.
      • Perez N.
      • Sumby P.
      The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript.

      Quantitative RT-PCR Analysis

      GAS strains were grown in triplicate at 37°C with 5% CO2 in THY to ME growth phase, as previously described.
      • Zhu L.
      • Olsen R.J.
      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      RNAprotect Bacteria Reagent was added at a 2:1 ratio, and then the cells were lysed by ballistic disintegration (FastPrep-96 instrument and lysing matrix B). Standard methods were used to extract RNA (RNeasy kit). The RNA was converted into cDNA using Superscript III Reverse Transcriptase (Invitrogen). Quantitative RT-PCR was conducted using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and an ABI 7500 Fast System instrument (Life Technologies, Carlsbad, CA). The sequences of the TaqMan primers and probes are listed in Table 1. Each experiment was performed with three biological replicates and two technical replicates. Transcript levels were measured relative to the constitutively expressed gene tufA.
      • Sanson M.
      • Makthal N.
      • Flores A.R.
      • Olsen R.J.
      • Musser J.M.
      • Kumaraswami M.
      Adhesin competence repressor (AdcR) from Streptococcus pyogenes controls adaptive responses to zinc limitation and contributes to virulence.

      Animal Infection Models

      Mouse Model of Necrotizing Myositis

      Mouse necrotizing fasciitis/myositis studies were performed, as previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Zhu L.
      • Olsen R.J.
      • Lee J.D.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Contribution of secreted NADase and streptolysin O to the pathogenesis of epidemic serotype M1 Streptococcus pyogenes infections.
      • Beres S.B.
      • Kachroo P.
      • Nasser W.
      • Olsen R.J.
      • Zhu L.
      • Flores A.R.
      • de la Riva I.
      • Paez-Mayorga J.
      • Jimenez F.E.
      • Cantu C.
      • Vuopio J.
      • Jalava J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Corander J.
      • Fittipaldi N.
      • Di Luca M.C.
      • Petrelli D.
      • Vitali L.A.
      • Raiford A.
      • Jenkins L.
      • Musser J.M.
      Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes.
      • Olsen R.J.
      • Sitkiewicz I.
      • Ayeras A.A.
      • Gonulal V.E.
      • Cantu C.
      • Beres S.B.
      • Green N.M.
      • Lei B.
      • Humbird T.
      • Greaver J.
      • Chang E.
      • Ragasa W.P.
      • Montgomery C.A.
      • Cartwright Jr., J.
      • McGeer A.
      • Low D.E.
      • Whitney A.R.
      • Cagle P.T.
      • Blasdel T.L.
      • DeLeo F.R.
      • Musser J.M.
      Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.
      Immunocompetent 4-week–old female CD1 mice (Envigo Laboratories, Houston, TX) were randomly assigned to treatment groups and inoculated in the right lower hind limb with 5 × 108 colony-forming units of the indicated bacterial strain suspended in 100 μL phosphate-buffered saline (n = 40 mice per strain). Mice were monitored at least once daily, and near-mortality was determined using NIH's Guide for the Care and Use of Laboratory Animals.
      Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council
      Guide for the Care and Use of Laboratory Animals: Eighth Edition.
      Survival was compared by the log-rank test. For gross and histologic evaluation, mice (n = 3 mice per strain) were sacrificed on day 1 after inoculation and the limbs were processed using standard methods.
      • Olsen R.J.
      • Sitkiewicz I.
      • Ayeras A.A.
      • Gonulal V.E.
      • Cantu C.
      • Beres S.B.
      • Green N.M.
      • Lei B.
      • Humbird T.
      • Greaver J.
      • Chang E.
      • Ragasa W.P.
      • Montgomery C.A.
      • Cartwright Jr., J.
      • McGeer A.
      • Low D.E.
      • Whitney A.R.
      • Cagle P.T.
      • Blasdel T.L.
      • DeLeo F.R.
      • Musser J.M.
      Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.

      NHP Model of Necrotizing Myositis

      A well-described NHP model of necrotizing fasciitis/myositis was used.
      • Zhu L.
      • Olsen R.J.
      • Nasser W.
      • Beres S.B.
      • Vuopio J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      • Olsen R.J.
      • Sitkiewicz I.
      • Ayeras A.A.
      • Gonulal V.E.
      • Cantu C.
      • Beres S.B.
      • Green N.M.
      • Lei B.
      • Humbird T.
      • Greaver J.
      • Chang E.
      • Ragasa W.P.
      • Montgomery C.A.
      • Cartwright Jr., J.
      • McGeer A.
      • Low D.E.
      • Whitney A.R.
      • Cagle P.T.
      • Blasdel T.L.
      • DeLeo F.R.
      • Musser J.M.
      Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      Briefly, NHPs (n = 8) were sedated and inoculated at a uniform depth with 1 × 109 colony-forming units/kg in the left or right quadriceps muscle (n = 4 limbs per strain). Animals were observed and necropsied 24 hours after infection.
      All animal experiments were approved by the Institutional Animal Care and Use Committee of the Houston Methodist Research Institute (Houston, TX; AUP-1217-0058 and AUP-0318-0016). Sample sizes for each experiment were determined with a power calculation.

      Statistical Analysis

      All statistical analyses were performed using Prism 7 (GraphPad Software Inc., La Jolla, CA) with three biological replicates, and P < 0.05 was considered statistically significant.

      Data Availability

      The serotype M28 RNA-seq sequence data have been deposited at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/bioproject; accession number PRJNA540250).

      Results

      Naturally Occurring Polymorphisms in rocA Are Associated with Altered Transcriptomes in Serotype M28 GAS Strains

      Whole genome sequence analysis of 2101 serotype M28 GAS strains recovered from large, comprehensive, population-based collections of human invasive infections identified 29 unique polymorphisms in rocA in 48 strains (Figure 1A and Supplemental Table S1).
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      Unlike other GAS serotypes previously investigated,
      • Beres S.B.
      • Kachroo P.
      • Nasser W.
      • Olsen R.J.
      • Zhu L.
      • Flores A.R.
      • de la Riva I.
      • Paez-Mayorga J.
      • Jimenez F.E.
      • Cantu C.
      • Vuopio J.
      • Jalava J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Corander J.
      • Fittipaldi N.
      • Di Luca M.C.
      • Petrelli D.
      • Vitali L.A.
      • Raiford A.
      • Jenkins L.
      • Musser J.M.
      Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes.
      • Nasser W.
      • Beres S.B.
      • Olsen R.J.
      • Dean M.A.
      • Rice K.A.
      • Long S.W.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Vuopio J.
      • Raisanen K.
      • Caugant D.A.
      • Steinbakk M.
      • Low D.E.
      • McGeer A.
      • Darenberg J.
      • Henriques-Normark B.
      • Van Beneden C.A.
      • Hoffmann S.
      • Musser J.M.
      Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences.
      • Fittipaldi N.
      • Beres S.B.
      • Olsen R.J.
      • Kapur V.
      • Shea P.R.
      • Watkins M.E.
      • Cantu C.C.
      • Laucirica D.R.
      • Jenkins L.
      • Flores A.R.
      • Lovgren M.
      • Ardanuy C.
      • Linares J.
      • Low D.E.
      • Tyrrell G.J.
      • Musser J.M.
      Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.
      the number of rocA allelic variants in serotype M28 GAS was greater than expected by chance (P < 0.01, Fisher exact test).
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      As a first approach to understanding the role of naturally occurring rocA polymorphisms in serotype M28 GAS biology, RNA-seq analysis was performed on all 48 clinical isolates with naturally occurring rocA polymorphisms, an isogenic rocA deletion mutant strain (ΔrocA),
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      and four phylogenetically matched WT strains. The phylogenetically matched WT strains were included in the RNA-seq analysis to normalize for genetic differences (besides rocA sequence) among the naturally occurring clinical isolates that, in principle, may alter the transcriptome (Supplemental Table S1).
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      Gene expression analysis was conducted at ME (OD600 = 0.5) and ES (OD600 = 1.65) growth phases, as previously described.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      Principal component analysis revealed that, in general, polymorphisms in rocA result in two distinct transcriptomes at ME growth phase (Figure 1B). One rocA transcriptome group was allied with the phylogenetically matched WT strains (cluster 1; 21/48 rocA polymorphism strains). The second rocA transcriptome group had rocA mutant strains with substantially altered transcriptomes that are more closely allied with the isogenic ΔrocA deletion mutant strain (cluster 2; 26/48 rocA polymorphism strains). One clinical isolate with a mutation in the mga regulatory gene had an outlier transcriptome (cluster 3) (Supplemental Table S1).
      • Sanson M.
      • O'Neill B.E.
      • Kachroo P.
      • Anderson J.R.
      • Flores A.R.
      • Valson C.
      • Cantu C.C.
      • Makthal N.
      • Karmonik C.
      • Fittipaldi N.
      • Kumaraswami M.
      • Musser J.M.
      • Olsen R.J.
      A naturally occurring single amino acid replacement in multiple gene regulator of group A streptococcus significantly increases virulence.
      • Sanson M.
      • Makthal N.
      • Gavagan M.
      • Cantu C.
      • Olsen R.J.
      • Musser J.M.
      • Kumaraswami M.
      Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.
      • Ribardo D.A.
      • McIver K.S.
      Defining the Mga regulon: comparative transcriptome analysis reveals both direct and indirect regulation by Mga in the group A streptococcus.
      More important, no naturally occurring rocA polymorphism strain had a transcriptome identical to its phylogenetically matched WT strain. That is, each rocA allele in the clinical isolates resulted in a unique transcriptome at ME growth phase.
      At ES growth phase, principal component analysis revealed four different transcriptome clusters (Figure 1C). Unlike with ME growth phase (Supplemental Figure S1), each cluster was heavily influenced by the presence of mutations in global gene regulators other than rocA (Figure 1C). Cluster 1 strains were rocA polymorphism strains, including the isogenic ΔrocA deletion mutant strain, that were allied with the WT strains. Cluster 2 strains had mutations in covRS, cluster 3 strains had mutations in ropB, and cluster 4 had the single strain with a mutation in mga. Consistent with the ME growth phase data, no naturally occurring rocA polymorphism strain had a transcriptome identical to its phylogenetically matched WT strain.
      A list of each strain's rocA polymorphism, transcriptome cluster at ME and ES growth phases, and genes with significantly altered transcript levels compared with its phylogenetically matched WT strain (absolute transcript fold change ≥ 1.5 and P < 0.05 after Wald test with Bonferroni correction for multiple comparisons) is provided in Supplemental Tables S2 and S3.

      Selection and Construction of Isogenic rocA Polymorphism Strains

      Because the clinical isolates with naturally occurring rocA polymorphisms may have mutations in other genes that also influence the global transcriptome (as evidenced by the ES transcriptome data) (Figure 1C and Supplemental Table S1), five rocA polymorphisms were selected for further characterization by generating isogenic mutant strains. The alleles were selected using multiple criteria, including the following: i) the nature of the polymorphism, with an emphasis on amino acid alterations (rather than protein truncations) in the predicted functional domains of RocA (Figure 1A)
      • Jain I.
      • Miller E.W.
      • Danger J.L.
      • Pflughoeft K.J.
      • Sumby P.
      RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus.
      ; ii) the predicted effect of the RocA polymorphism based on the global transcriptome of the naturally occurring clinical isolates (Figure 1, B and C); and iii) the independent recurrence of the same polymorphism in multiple strains or the repeated presence of polymorphisms in the same codon (Figure 1A). Based mainly on these criteria, five RocA variants were selected for analysis: -9del(6nt) + H60Y [hereinafter referred to as variable number tandem repeat (VNTR) + H60Y], P97L, G184W, R258K, and T442P (Figure 1D).
      The VNTR + H60Y RocA variant was selected because its respective clinical isolate is the only naturally occurring strain to have two polymorphisms in rocA (Figure 1A). The VNTR sequence has been previously studied in serotype M89 GAS.
      • Zhu L.
      • Olsen R.J.
      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      In M89 strains, the VNTR deletion results in a lack of RocA protein translation due to a presumed loss of the ribosomal binding site.
      • Zhu L.
      • Olsen R.J.
      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      The P97L RocA variant was selected because codon 97 occurs in an inferred transmembrane domain (Figure 1D), and the RocA P97L clinical isolate had a rocA deletion-like transcriptome at ME growth phase (Supplemental Table S2). The G184W RocA variant was selected because codon 184 occurs in a different inferred transmembrane domain (Figure 1D) and is also polymorphic in serotype M89 GAS (Supplemental Figure S2). The R258K RocA variant was selected because codon 258 occurs in the H box of the putative HK domain (Figure 1D).
      • Biswas I.
      • Scott J.R.
      Identification of rocA, a positive regulator of covR expression in the group A streptococcus.
      The T442P RocA variant was selected because multiple clinical isolates (n = 9) acquired the polymorphism, a second polymorphism also occurs in codon 442 (T442I) (Figure 1A), and the T442P clinical isolates had substantially altered transcriptomes compared with their phylogenetically matched WT strains at ME growth phase (Supplemental Table S2). The isogenic rocA mutant strains were generated using allelic exchange in parental WT strain MGAS28426, as previously described.
      • Zhu L.
      • Olsen R.J.
      • Nasser W.
      • Beres S.B.
      • Vuopio J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      MGAS28426 was selected as it is genetically representative of serotype M28 GAS strains
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      and contains a WT allele for all major global transcription regulatory genes. It is also the parental strain used for the isogenic rocA deletion mutant strain
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      and has been previously used in animal infection studies.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      Whole genome sequencing of each isogenic rocA mutant strain confirmed the absence of spurious mutations.
      To determine whether rocA polymorphisms alter the growth phenotype of serotype M28 GAS, the parental WT strain, isogenic rocA deletion mutant strain (ΔrocA), and five isogenic rocA mutant strains were grown in nutrient-rich THY. The growth curves of the G184W and R258K isogenic mutant strains were indistinguishable from the parental WT strain (Figure 2A). In comparison, the isogenic ΔrocA deletion mutant strain and VNTR + H60Y, P97L, and T442P isogenic mutant strains had a decreased lag phase (Figure 2A). However, the slope of the growth curves did not differ significantly.
      Figure thumbnail gr2
      Figure 2Polymorphisms in rocA significantly alter the GAS transcriptome. A: Growth curve of the parental wild-type (WT) and isogenic rocA mutant strains in nutrient-rich THY. B and C: Principal component analysis (PCA) of the RNA-sequencing data generated from the parental WT strain and isogenic rocA mutant strains at midexponential (ME; B) and early-stationary (ES; C) growth phases. Each growth phase is represented by two PCA plots that are rotated approximately 45 degrees about the PC2 axis. D and E: Comparison of differentially expressed genes in the isogenic ΔrocA deletion mutant strain and isogenic variable number tandem repeat (VNTR) + H60Y, P97L, and T442P mutant strains at ME (D) and ES (E) growth phases.
      • Chen H.
      • Boutros P.C.
      VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R.

      Polymorphisms in rocA Result in an Altered Transcriptome in M28 GAS Strains

      To test the hypothesis that naturally occurring rocA polymorphisms alter global gene transcript levels in serotype M28 GAS, RNA-seq analysis was performed using the parental WT strain, isogenic ΔrocA deletion mutant strain, and five isogenic rocA mutant strains grown to ME and ES growth phases. Principal component analysis revealed that each rocA polymorphism conferred a unique transcriptome that varied from the WT strain to different extents (Figure 2, B and C). Whereas the G184W and R258K isogenic mutant strains had transcriptomes more similar to the parental WT strain, the VNTR + H60Y, P97L, and T442P isogenic mutant strain transcriptomes (hereinafter referred to collectively as the deletion-like strains) were allied with the isogenic ΔrocA deletion mutant strain at both ME and ES growth phases (Figure 2, B and C).
      Between 1 and 319 genes (0.05% to 17.48% of GAS genes) per strain were found to have significantly altered transcript levels at both ME and ES growth phases relative to the parental WT strain (absolute fold change ≥ 1.5 and P < 0.05 after Wald test with Bonferroni correction for multiple comparisons). The R258K isogenic mutant strain had 1 and 5 significantly altered transcript levels at ME and ES growth phases, respectively. Similarly, the G184W isogenic mutant strain had 22 and 25 significantly altered transcripts at ME and ES growth phases, respectively. In comparison, the isogenic ΔrocA deletion mutant strain and three deletion-like strains (VNTR + H60Y, P97L, and T442P isogenic mutant strains) had a common set of 130 and 139 genes at ME and ES growth phases, respectively, that were significantly differentially regulated (Figure 2, D and E).
      • Chen H.
      • Boutros P.C.
      VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R.
      Additional genes were identified as significantly differentially expressed in some, but not all, of the deletion-like strains (Figure 2, D and E). Genes with significantly altered transcript levels in each strain are listed in Supplemental Tables S4 and S5.

      Polymorphisms in rocA Confer Two Distinct Responses to Acid Stress in M28 GAS

      RNA-seq analysis showed that genes implicated in acidic stress response had altered transcript levels in the isogenic ΔrocA deletion mutant and isogenic rocA mutant strains (Supplemental Tables S4 and S5). For example, the arcABCD, ciaH, and spxA2 genes were significantly differentially regulated compared with the parental WT strain.
      • Tatsuno I.
      • Isaka M.
      • Okada R.
      • Zhang Y.
      • Hasegawa T.
      Relevance of the two-component sensor protein CiaH to acid and oxidative stress responses in Streptococcus pyogenes.
      • Cusumano Z.T.
      • Watson Jr., M.E.
      • Caparon M.G.
      Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity.
      • Cusumano Z.T.
      • Caparon M.G.
      Citrulline protects Streptococcus pyogenes from acid stress using the arginine deiminase pathway and the F1Fo-ATPase.
      • Port G.C.
      • Cusumano Z.T.
      • Tumminello P.R.
      • Caparon M.G.
      SpxA1 and SpxA2 act coordinately to fine-tune stress responses and virulence in Streptococcus pyogenes.
      Increased resistance to acid stress, a physiological condition present in a developing purulent lesion,
      • Nekoofar M.H.
      • Namazikhah M.S.
      • Sheykhrezae M.S.
      • Mohammadi M.M.
      • Kazemi A.
      • Aseeley Z.
      • Dummer P.M.H.
      pH of pus collected from periapical abscesses.
      • Bryant R.E.
      • Mazza J.A.
      Effect of the abscess environment on the antimicrobial activity of ciprofloxacin.
      • Wiese K.G.
      Electrolyte concentration, real and osmotic pressure in abscesses.
      is one possible advantage for GAS to inactivating RocA in vivo. To test the hypothesis that polymorphisms in rocA alter GAS growth when exposed to acidic stress, the parental WT strain, isogenic ΔrocA deletion mutant strain, and five isogenic rocA mutant strains were grown in THY buffered with 2-(N-morpholino)ethanesulfonic acid (pH = 6.0). Consistent with our hypothesis, the isogenic ΔrocA deletion mutant strain and three deletion-like strains (VNTR + H60Y, P97L, and T442P isogenic mutant strains) grown in acidic conditions [2-(N-morpholino)ethanesulfonic acid; pH = 6.0] had a shortened lag phase and an increased slope of the exponential phase compared with the parental WT strain (Figure 3A). In contrast, the G184W and R258K isogenic mutant strains did not significantly differ in overall growth from the parental WT strain (Figure 3A). Taken together, the data show that polymorphisms in rocA alter the growth phenotype of M28 GAS strains in acidic conditions and suggest they contribute to altered molecular pathogenesis during human invasive infections.
      Figure thumbnail gr3
      Figure 3Polymorphisms in rocA result in an altered virulence phenotype in vitro. A: Growth curve of the parental wild-type (WT) and isogenic rocA polymorphism strains in THY buffered with 0.1 mol/L 2-(N-morpholino)ethanesulfonic acid (pH = 6.0). B: Western immunoblot analysis of NAD+-glycohydrolase (SPN) and streptolysin O (SLO). C: SPN activity. D: SLO activity. E: Serum opacity factor (SOF) activity. Data are expressed as means ± SD (CE). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus WT (one-way analysis of variance with Dunnett multiple comparisons test). VNTR, variable number tandem repeat.

      Polymorphisms in rocA Confer Different Virulence Factor Expression and Enzymatic Activity Profiles in M28 GAS Strains

      Consistent with our previously published isogenic ΔrocA deletion mutant strain transcriptome data,
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      several proven and putative virulence factors had significantly altered transcript levels in the isogenic rocA mutant strains (Supplemental Table S6). Selected virulence factors include the Mga regulon virulence factors
      • Sanson M.
      • O'Neill B.E.
      • Kachroo P.
      • Anderson J.R.
      • Flores A.R.
      • Valson C.
      • Cantu C.C.
      • Makthal N.
      • Karmonik C.
      • Fittipaldi N.
      • Kumaraswami M.
      • Musser J.M.
      • Olsen R.J.
      A naturally occurring single amino acid replacement in multiple gene regulator of group A streptococcus significantly increases virulence.
      • Sanson M.
      • Makthal N.
      • Gavagan M.
      • Cantu C.
      • Olsen R.J.
      • Musser J.M.
      • Kumaraswami M.
      Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.
      • Ribardo D.A.
      • McIver K.S.
      Defining the Mga regulon: comparative transcriptome analysis reveals both direct and indirect regulation by Mga in the group A streptococcus.
      : sclA,
      • Lukomski S.
      • Nakashima K.
      • Abdi I.
      • Cipriano V.J.
      • Ireland R.M.
      • Reid S.D.
      • Adams G.G.
      • Musser J.M.
      Identification and characterization of the scl gene encoding a group A Streptococcus extracellular protein virulence factor with similarity to human collagen.
      fba,
      • Terao Y.
      • Kawabata S.
      • Kunitomo E.
      • Murakami J.
      • Nakagawa I.
      • Hamada S.
      Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator.
      • Rouchon C.N.
      • Ly A.T.
      • Noto J.P.
      • Luo F.
      • Lizano S.
      • Bessen D.E.
      Incremental contributions of FbaA and other impetigo-associated surface proteins to fitness and virulence of a classical group A streptococcal skin strain.
      scpA,
      • Wexler D.E.
      • Chenoweth D.E.
      • Cleary P.P.
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      Many group A streptococcal strains express two different immunoglobulin-binding proteins, encoded by closely linked genes: characterization of the proteins expressed by four strains of different M-type.
      emm,
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      The streptococcal M protein: a highly versatile molecule.
      mrp,
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      sfbX,
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      Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX.
      and sof (encoding SOF).
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      Opacification domain of serum opacity factor inhibits beta-hemolysis and contributes to virulence of Streptococcus pyogenes.
      Other differentially expressed genes are CovRS regulated virulence factors,
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      A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.
      such as nga (encoding SPN),
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      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      slo (encoding SLO),
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      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      spyCEP,
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      Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes.
      mac,
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      IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.
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      The intrinsic immunoglobulin G endopeptidase activity of streptococcal Mac-2 proteins implies a unique role for the enzymatically impaired Mac-2 protein of M28 serotype strains.
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      Opsonophagocytosis-inhibiting Mac protein of group A Streptococcus: identification and characterisitcs of two gene complexes.
      sse,
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      Group A Streptococcus secreted esterase hydrolyzes platelet-activating factor to impede neutrophil recruitment and facilitate innate immune evasion.
      ska (encoding SKA),
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      • Malke H.
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      Heterogeneity of the streptokinase gene in group A streptococci.
      and speB (encoding SpeB)
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      The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.
      (Supplemental Table S6). To measure the phenotypic effect of differential transcript levels associated with each rocA polymorphism, in vitro virulence factor assays were performed. The isogenic ΔrocA deletion mutant strain and three deletion-like strains (VNTR + H60Y, P97L, and T442P isogenic mutant strains) had increased expression of secreted immunoreactive SPN and SLO protein (Figure 3B) and significantly increased SPN NAD+-glycohydrolase, SLO cytotoxin, and SOF secreted activity (Figure 3, C–E), consistent with the RNA-seq data. In comparison, the R258K isogenic mutant strain had WT-like virulence factor expression and activity (Figure 3). In contrast, but also consistent with the RNA-seq data (Supplemental Table S6), the G184W isogenic mutant strain had decreased expression of secreted immunoreactive SPN and SLO protein (Figure 3B) and significantly decreased secreted activity of SPN, SLO, and SOF (Figure 3, C–E). No difference in secreted SpeB protease activity was observed among the isogenic mutant strains compared with the parental WT strain (Supplemental Figure S3). Thus, different polymorphisms in rocA result in different in vitro virulence phenotypes.

      Polymorphisms in rocA Confer Different Secreted Streptokinase Activities

      In addition to the aforementioned virulence factors, ska, the gene encoding SKA,
      • Huang T.-T.
      • Malke H.
      • Ferretti J.J.
      Heterogeneity of the streptokinase gene in group A streptococci.
      was differentially regulated in the isogenic rocA polymorphism strains (Supplemental Table S6). At ES growth phase, ska had the largest range of transcript levels among the common set of genes differentially expressed by the isogenic ΔrocA deletion mutant and deletion-like strains (VNTR + H60Y, P97L, and T442P isogenic mutant strains) (Supplemental Table S5). The ska gene was also significantly differentially expressed at ME growth phase only by the T442P isogenic mutant strain (Supplemental Table S4). To determine whether polymorphisms in rocA decrease secreted SKA activity, an in vitro assay was performed. At ME growth phase, the isogenic ΔrocA deletion mutant strain and all five isogenic rocA mutant strains had significantly decreased secreted SKA activity compared with the parental WT strain (Figure 4A). Whereas the G184W and R258K isogenic mutant strains (WT-like transcriptomes) had secreted SKA activity more similar to the WT strain, the three deletion-like strains (VNTR + H60Y, P97L, and T442P isogenic mutant strains) had markedly lower secreted SKA activity (Figure 4A). Unexpectedly, the P97L isogenic mutant strain had an intermediate level of secreted SKA activity, and the T442P isogenic mutant strain had very low secreted SKA activity (Figure 4A). That is, the P97L and T442P isogenic mutant strains had significantly increased and decreased secreted SKA activity, respectively, compared with the isogenic ΔrocA deletion mutant strain. Similar data were observed at ES growth phase (Figure 4B).
      Figure thumbnail gr4
      Figure 4Polymorphisms in rocA result in decreased ska transcript levels and streptokinase (SKA) activity, in part, due to altered fasX regulation. A and B: SKA activity at midexponential (ME; A) and early-stationary (ES; B) growth phases. C: Quantitative RT-PCR analysis of fasX at ME growth phase. The log2 fold changes relative to tufA are shown. Data are expressed as means ± SD (AC). **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the parental WT strain (one-way analysis of variance with Tukey multiple comparisons test); P < 0.05, †††P < 0.001, and ††††P < 0.0001 versus the isogenic ΔrocA deletion mutant strain (one-way analysis of variance with Tukey multiple comparisons test). VNTR, variable number tandem repeat; WT, wild type.
      The ska gene is regulated by the CovRS and FasBCA/fasX systems.
      • Ramirez-Pena E.
      • Trevino J.
      • Liu Z.
      • Perez N.
      • Sumby P.
      The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript.
      • Churchward G.
      • Bates C.
      • Gusa A.A.
      • Stringer V.
      • Scott J.R.
      Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.
      We hypothesized that fasX may contribute to the unexpectedly low ska transcripts and SKA activity in the T442P strain. fasX encodes a small RNA that is regulated by FasBCA and enhances ska transcript stability.
      • Ramirez-Pena E.
      • Trevino J.
      • Liu Z.
      • Perez N.
      • Sumby P.
      The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript.
      Because our RNA-seq protocol cannot reliably measure transcripts of small RNAs, such as fasX, quantitative RT-PCR analysis of fasX was performed for the parental WT strain, isogenic ΔrocA deletion mutant strain, and isogenic rocA mutant strains at ME growth phase. As expected, the T442P isogenic mutant strain had significantly decreased expression of fasX compared with the parental WT strain (Figure 4C). Thus, differences in secreted SKA activity among the rocA polymorphism strains may, in part, be explained by altered expression of fasX.

      Polymorphisms in rocA Confer Two Distinct Virulence Phenotypes in a Mouse Model of Necrotizing Myositis

      To determine whether polymorphisms in rocA result in altered virulence, the parental WT strain, isogenic ΔrocA deletion mutant strain, and five isogenic rocA mutant strains were compared using a well-established necrotizing myositis mouse model.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
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      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      • Beres S.B.
      • Kachroo P.
      • Nasser W.
      • Olsen R.J.
      • Zhu L.
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      Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes.
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      • Musser J.M.
      Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.
      Compared with the parental WT strain, the ΔrocA, VNTR + H60Y, and T442P isogenic mutant strains caused significantly increased near mortality (Figure 5A) and larger lesions with more tissue destruction (Figure 5B). Isogenic mutant strains containing either the G184W or R258K amino acid replacements (WT-like transcriptomes) did not differ significantly in virulence from the parental WT strain (Figure 5A). Unexpectedly, the mouse virulence of the P97L isogenic mutant strain, which has a rocA deletion-like transcriptome, did not significantly differ from the parental WT strain (Figure 5A).
      Figure thumbnail gr5
      Figure 5Polymorphisms in rocA result in altered virulence in mouse and nonhuman primate (NHP) models of necrotizing myositis. A: Kaplan-Meier survival curve for mice infected in the right hind limb with the indicated strain. Log-rank test was performed. B: Representative microscopic lesions of the infected limb on postinoculation day 1. The necrotic lesions are encompassed by boxed areas. Hematoxylin and eosin staining was used. C: Lesion volume of NHPs infected in the quadriceps muscle with the indicated strain. U-test was performed. D: Representative microscopic lesions of the infected muscle. The necrotic lesions are encompassed by boxed areas. Hematoxylin and eosin staining was used. n = 40 mice per strain (A); n = 4 limbs per strain (C). *P < 0.05 versus wild type (WT). Original magnification, ×4 (B and D). VNTR, variable number tandem repeat.
      To begin to identify the possible molecular basis of the decreased virulence phenotype of the P97L strain, the RNA-seq data were reexamined. Compared with the other deletion-like transcriptome strains (ΔrocA, VNTR + H60Y, and T442P isogenic mutant strains), the P97L isogenic mutant strain had 10 genes with significantly altered transcript levels (Supplemental Table S7). More important, compared with the other deletion-like transcriptome strains, the isogenic mutant with the P97L amino acid change had significantly decreased transcript levels of four genes (slo, prsA, mac, and sclA) encoding proven virulence factors.
      • Zhu L.
      • Olsen R.J.
      • Nasser W.
      • Beres S.B.
      • Vuopio J.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      A molecular trigger for intercontinental epidemics of group A Streptococcus.
      • Olsen R.J.
      • Sitkiewicz I.
      • Ayeras A.A.
      • Gonulal V.E.
      • Cantu C.
      • Beres S.B.
      • Green N.M.
      • Lei B.
      • Humbird T.
      • Greaver J.
      • Chang E.
      • Ragasa W.P.
      • Montgomery C.A.
      • Cartwright Jr., J.
      • McGeer A.
      • Low D.E.
      • Whitney A.R.
      • Cagle P.T.
      • Blasdel T.L.
      • DeLeo F.R.
      • Musser J.M.
      Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.
      • Lukomski S.
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      Identification and characterization of the scl gene encoding a group A Streptococcus extracellular protein virulence factor with similarity to human collagen.
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      Blocking of experimental arthritis by cleavage of IgG antibodies in vivo.
      Thus, altered expression of slo, prsA, mac, and sclA may explain, in part, the unexpectedly lower virulence phenotype of the P97L isogenic mutant strain.

      Polymorphisms in rocA Result in Altered Virulence in a NHP Model of Necrotizing Myositis

      GAS is a human-specific pathogen, and some virulence factors have specificity for human and NHP molecules.
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      To unambiguously demonstrate that polymorphisms in rocA contribute to serotype M28 GAS virulence, four strains were compared using an NHP model of necrotizing myositis. We hypothesized that the isogenic ΔrocA deletion mutant and T442P (deletion-like) isogenic mutant strains are significantly more virulent than the parental WT or G184W (WT-like) isogenic mutant strains. Consistent with our hypothesis, the isogenic ΔrocA deletion mutant and T442P isogenic mutant strains caused significantly larger lesions (Figure 5C) with more tissue destruction (Figure 5D).

      Discussion

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      A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.
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      A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.
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      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      Using several different GAS serotype strains, it has been demonstrated that inactivation of CovRS or RocA leads to significantly increased virulence in mice, NHPs, and human patients.
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      A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.
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      Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis.
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      • Ramalinga A.B.
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      • Shelburne S.A.
      • Sumby P.
      Regulatory rewiring confers serotype-specific hyper-virulence in the human pathogen group A Streptococcus.
      • Zhu L.
      • Olsen R.J.
      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      • Feng W.
      • Minor D.
      • Liu M.
      • Li J.
      • Ishaq S.L.
      • Yeoman C.
      • Lei B.
      Null mutations of group A Streptococcus orphan kinase RocA: selection in mouse infection and comparison with CovS mutations in alteration of in vitro and in vivo protease SpeB expression and virulence.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Sarkar P.
      • Danger J.L.
      • Jain I.
      • Meadows L.A.
      • Beam C.
      • Medicielo J.
      • Burgess C.
      • Musser J.M.
      • Sumby P.
      Phenotypic variation in the group A Streptococcus due to natural mutation in the accessory protein-encoding gene rocA.
      • Sugareva V.
      • Arlt R.
      • Fiedler T.
      • Riani C.
      • Podbielski A.
      • Kreikemeyer B.
      Serotype- and strain-dependent contribution of the sensor kinase CovS of the CovRS two-component system to Streptococcus pyogenes pathogenesis.
      • Bao Y.J.
      • Liang Z.
      • Mayfield J.A.
      • Lee S.W.
      • Ploplis V.A.
      • Castellino F.J.
      CovRS-regulated transcriptome analysis of a hypervirulent M23 strain of group A Streptococcus pyogenes provides new insights into virulence determinants.
      • Levin J.C.
      • Wessels M.R.
      Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus.
      • Tatsuno I.
      • Okada R.
      • Zhang Y.
      • Isaka M.
      • Hasegawa T.
      Partial loss of CovS function in Streptococcus pyogenes causes severe invasive disease.
      • Flores A.R.
      • Sahasrabhojane P.
      • Saldana M.
      • Galloway-Pena J.
      • Olsen R.J.
      • Musser J.M.
      • Shelburne S.A.
      Molecular characterization of an invasive phenotype of group A Streptococcus arising during human infection using whole genome sequencing of multiple isolates from the same patient.
      • Yoshida H.
      • Ishigaki Y.
      • Takizawa A.
      • Moro K.
      • Kishi Y.
      • Takahashi T.
      • Matsui H.
      Comparative genomics of the mucoid and nonmucoid strains of Streptococcus pyogenes, isolated from the same patient with streptococcal meningitis.
      • Ikebe T.
      • Matsumura T.
      • Nihonmatsu H.
      • Ohya H.
      • Okuno R.
      • Mitsui C.
      • Kawahara R.
      • Kameyama M.
      • Sasaki M.
      • Shimada N.
      • Ato M.
      • Ohnishi M.
      Spontaneous mutations in Streptococcus pyogenes isolates from streptococcal toxic shock syndrome patients play roles in virulence.
      Recent whole genome sequence analysis of large, comprehensive, population-based collections of serotype M28 GAS strains demonstrated higher numbers of polymorphisms in rocA than expected.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Kachroo P.
      • Eraso J.M.
      • Beres S.B.
      • Olsen R.J.
      • Zhu L.
      • Nasser W.
      • Bernard P.E.
      • Cantu C.C.
      • Ojeda Saavedra M.
      • Arredondo M.J.
      • Strope B.
      • Do H.
      • Kumaraswami M.
      • Vuopio J.
      • Grondahl-Yli-Hannuksela K.
      • Kristinsson K.G.
      • Gottfredsson M.
      • Pesonen M.
      • Pensar J.
      • Davenport E.R.
      • Clark A.G.
      • Corander J.
      • Caugant D.A.
      • Gaini S.
      • Magnussen M.D.
      • Kubiak S.L.
      • Nguyen H.A.T.
      • Long S.W.
      • Porter A.R.
      • DeLeo F.R.
      • Musser J.M.
      Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.
      Herein, we investigated the effect of different naturally occurring polymorphisms in rocA on serotype M28 GAS gene expression, virulence factor activity, and virulence in mice and NHPs (Figure 6). We discovered that each polymorphism resulted in either a subtly altered WT-like transcriptome or a substantially altered rocA deletion-like transcriptome (Figure 2). Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, these data suggest that naturally occurring polymorphisms in rocA contribute to the molecular pathogenesis of serotype M28 GAS invasive strains.
      Figure thumbnail gr6
      Figure 6Model of RocA contribution to the molecular pathogenesis of serotype M28 GAS. M28 GAS strains with a wild-type (WT) rocA gene or WT-like variant, such as R258K (red panel), have normal levels of RocA-regulated genes and a normal virulence phenotype. Mutation of RocA at codon 184 (brown panel) results in decreased transcript levels of some virulence factor genes but no change in virulence phenotype. In contrast, M28 GAS strains with a deletion of rocA or loss of RocA translation [variable number tandem repeat (VNTR) + H60Y; blue panel] have a substantially altered transcriptome that significantly increases virulence factor expression and virulence in mice and nonhuman primates. Similarly, mutation at codon 442 (purple panel) or codon 97 (orange panel) also results in a rocA deletion-like phenotype. CovR, control of virulence regulator; CovS, control of virulence sensor.
      Our laboratory and others have recently used transposon mutagenesis libraries to identify genes essential for GAS fitness, including serotype M28 GAS strains, in various anatomic niches.
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      • Le Breton Y.
      • Mistry P.
      • Valdes K.M.
      • Quigley J.
      • Kumar N.
      • Tettelin H.
      • McIver K.S.
      Genome-wide identification of genes required for fitness of group A streptococus in human blood.
      • Le Breton Y.
      • Belew A.T.
      • Freiberg J.A.
      • Sundar G.S.
      • Islam E.
      • Lieberman J.
      • Shirtliff M.E.
      • Tettelin H.
      • El-Sayed N.M.
      • McIver K.S.
      Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.
      • Zhu L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Olsen R.J.
      • Beres S.B.
      • Musser J.M.
      Novel genes required for the fitness of Streptococcus pyogenes in human saliva.
      Consistent with the data reported herein, transposon mutagenesis studies have shown rocA inactivation to significantly increase GAS fitness in mouse s.c. and NHP necrotizing myositis infection models.
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      • Le Breton Y.
      • Belew A.T.
      • Freiberg J.A.
      • Sundar G.S.
      • Islam E.
      • Lieberman J.
      • Shirtliff M.E.
      • Tettelin H.
      • El-Sayed N.M.
      • McIver K.S.
      Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.
      The RNA-seq data demonstrate that rocA polymorphisms and rocA gene deletion significantly alter expression of many proven and putative virulence factors (Figure 3 and Supplemental Table S6).
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      As a result, rocA polymorphisms significantly increase M28 GAS virulence (Figure 5). Many RocA regulated genes are also individually predicted by the transposon mutagenesis studies to be essential to GAS fitness (Supplemental Table S8).
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      • Le Breton Y.
      • Mistry P.
      • Valdes K.M.
      • Quigley J.
      • Kumar N.
      • Tettelin H.
      • McIver K.S.
      Genome-wide identification of genes required for fitness of group A streptococus in human blood.
      • Le Breton Y.
      • Belew A.T.
      • Freiberg J.A.
      • Sundar G.S.
      • Islam E.
      • Lieberman J.
      • Shirtliff M.E.
      • Tettelin H.
      • El-Sayed N.M.
      • McIver K.S.
      Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.
      • Zhu L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Olsen R.J.
      • Beres S.B.
      • Musser J.M.
      Novel genes required for the fitness of Streptococcus pyogenes in human saliva.
      For example, several transporters regulated by RocA alter GAS fitness. Deletion of a putative methionine transporter system (encoded by M28_Spy0263/0264/0265) and two putative export systems (encoded by M28_Spy0625/0626/0627 and M28_Spy1711/1712) results in decreased fitness in human saliva ex vivo and NHP muscle in vivo.
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      • Zhu L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Olsen R.J.
      • Beres S.B.
      • Musser J.M.
      Novel genes required for the fitness of Streptococcus pyogenes in human saliva.
      In addition, genes encoding several proven virulence factors regulated by RocA also contribute to GAS fitness in various anatomic niches (Supplemental Table S8).
      • Flores A.R.
      • Olsen R.J.
      • Cantu C.
      • Pallister K.B.
      • Guerra F.E.
      • Voyich J.M.
      • Musser J.M.
      Increased pilus production conferred by a naturally occurring mutation alters host-pathogen interaction in favor of carriage in Streptococcus pyogenes.
      • Tatsuno I.
      • Isaka M.
      • Okada R.
      • Zhang Y.
      • Hasegawa T.
      Relevance of the two-component sensor protein CiaH to acid and oxidative stress responses in Streptococcus pyogenes.
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      • Cusumano Z.T.
      • Watson Jr., M.E.
      • Caparon M.G.
      Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity.
      • Cusumano Z.T.
      • Caparon M.G.
      Citrulline protects Streptococcus pyogenes from acid stress using the arginine deiminase pathway and the F1Fo-ATPase.
      • Le Breton Y.
      • Mistry P.
      • Valdes K.M.
      • Quigley J.
      • Kumar N.
      • Tettelin H.
      • McIver K.S.
      Genome-wide identification of genes required for fitness of group A streptococus in human blood.
      • Le Breton Y.
      • Belew A.T.
      • Freiberg J.A.
      • Sundar G.S.
      • Islam E.
      • Lieberman J.
      • Shirtliff M.E.
      • Tettelin H.
      • El-Sayed N.M.
      • McIver K.S.
      Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.
      • Zhu L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Olsen R.J.
      • Beres S.B.
      • Musser J.M.
      Novel genes required for the fitness of Streptococcus pyogenes in human saliva.
      • Crotty Alexander L.E.
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      • Timmer A.M.
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      • von Kockritz-Blickwede M.
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      M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment.
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      Identification and characterization of serotype-specific variation in group A Streptococcus pilus expression.
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      D-alanylation of teichoic acids promotes group A Streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion.
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      Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes.
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      Regulation of glutamine and glutamate metabolism by GlnR and GlnA in Streptococcus pneumoniae.
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      Contribution of glutamine synthetase to the virulence of Streptococcus suis serotype 2.
      Furthermore, increased expression of nga and slo (Figure 3 and Supplemental Table S8) was recently implicated in the emergence and global dissemination of epidemic serotype M1 and M89 GAS strains.
      • Zhu L.
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      A molecular trigger for intercontinental epidemics of group A Streptococcus.
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      Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes.
      • Nasser W.
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      Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences.
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      Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events.
      • Zhu L.
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      Trading capsule for increased cytotoxin production: contribution to virulence of a newly emerged clade of emm89 Streptococcus pyogenes.
      Regulation of the ska gene by RocA in serotype M28 GAS is unique.
      • Bernard P.E.
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      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      Although rocA polymorphisms in other GAS serotype strains result in increased ska transcript and SKA protein levels, rocA polymorphisms in M28 GAS strains cause decreased ska transcript levels and secreted SKA activity (Supplemental Table S6 and Figure 4).
      • Miller E.W.
      • Danger J.L.
      • Ramalinga A.B.
      • Horstmann N.
      • Shelburne S.A.
      • Sumby P.
      Regulatory rewiring confers serotype-specific hyper-virulence in the human pathogen group A Streptococcus.
      • Bernard P.E.
      • Kachroo P.
      • Zhu L.
      • Beres S.B.
      • Eraso J.M.
      • Kajani Z.
      • Long S.W.
      • Musser J.M.
      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      • Jain I.
      • Miller E.W.
      • Danger J.L.
      • Pflughoeft K.J.
      • Sumby P.
      RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus.
      • Sarkar P.
      • Danger J.L.
      • Jain I.
      • Meadows L.A.
      • Beam C.
      • Medicielo J.
      • Burgess C.
      • Musser J.M.
      • Sumby P.
      Phenotypic variation in the group A Streptococcus due to natural mutation in the accessory protein-encoding gene rocA.
      The ska gene is regulated by the FasBCA/fasX and CovRS systems,
      • Kreikemeyer B.
      • Boyle M.D.P.
      • Buttaro B.A.
      • Heinemann M.
      • Podbielski A.
      Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologues to two-component-type regulators requires a small RNA molecule.
      • Danger J.L.
      • Cao T.N.
      • Cao T.H.
      • Sarkar P.
      • Trevino J.
      • Pflughoeft K.J.
      • Sumby P.
      The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype-specific targets.
      • Sumby P.
      • Whitney A.R.
      • Graviss E.A.
      • DeLeo F.R.
      • Musser J.M.
      Genome-wide analysis of group A streptococci reveals a mutation that modulates global phenotype and disease specificity.
      • Shelburne S.A.
      • Olsen R.J.
      • Suber B.
      • Sahasrabhojane P.
      • Sumby P.
      • Brennan R.G.
      • Musser J.M.
      A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.
      • Graham M.R.
      • Smoot L.M.
      • Lux Migiliaccio C.A.
      • Virtaneva K.
      • Sturdevant D.E.
      • Porcella S.F.
      • Federle M.J.
      • Adams G.J.
      • Scott J.R.
      • Musser J.M.
      Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.
      • Horstmann N.
      • Saldana M.
      • Sahasrabhojane P.
      • Yao H.
      • Su X.
      • Thompson E.
      • Koller A.
      • Shelburne 3rd, S.A.
      Dual-site phosphorylation of the control of virulence regulator impacts group A streptococcal global gene expression and pathogenesis.
      • Horstmann N.
      • Sahasrabhojane P.
      • Saldana M.
      • Ajami N.J.
      • Flores A.R.
      • Sumby P.
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      • Yao H.
      • Su X.
      • Thompson E.
      • Shelburne S.A.
      Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus.
      • Ramirez-Pena E.
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      • Liu Z.
      • Perez N.
      • Sumby P.
      The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript.
      • Churchward G.
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      • Stringer V.
      • Scott J.R.
      Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.
      and decreased fasX transcripts in the isogenic RocA T442P mutant strain likely resulted in a more severely decreased SKA activity phenotype (Figure 4). Our data suggest that RocA may interact, either directly or indirectly, with the FasBCA/fasX system to alter SKA activity. In addition, M28 GAS strains with a polymorphism in rocA have altered expression of gidA, a tRNA modifying enzyme that affects transcript levels of genes encoding several virulence factors, including ska (Supplemental Table S5).
      • Cho K.H.
      • Caparon M.G.
      tRNA modification by GidA/MnmE is necessary for Streptococcus pyogenes virulence: a new strategy to make live attenuated strains.
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      • Bailey J.R.
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      • Musser J.M.
      Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus.
      Interestingly, the transposon mutagenesis library screens found deletion of fasBCA to result in increased GAS fitness for serotype M28, but not M1, in NHP necrotizing myositis.
      • Zhu L.
      • Olsen R.J.
      • Beres S.B.
      • Eraso J.M.
      • Ojeda Saavedra M.
      • Kubiak S.L.
      • Cantu C.C.
      • Jenkins L.
      • Charbonneau A.R.L.
      • Waller A.S.
      • Musser J.M.
      Gene fitness landscape of group A streptococcus during necrotizing myositis.
      Thus, the RocA and FasBCA/fasX systems may have serotype-specific regulatory functions.
      Polymorphisms in RocA or rocA gene deletion results in significantly altered transcript levels of speB and hasABC (Supplemental Tables S2, S3, S4, and S5). The speB gene encodes a secreted cysteine protease and well-documented virulence factor.
      • Olsen R.J.
      • Raghuram A.
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      • Hartman M.H.
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      • Lee S.
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      • Valson C.
      • Flores A.R.
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      • Nasser W.
      • Musser J.M.
      The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.
      Although there were differences in speB transcript levels, there was no difference in secreted protease activity (Supplemental Figure S3), which may be due to complex regulation and activation of SpeB.
      • Olsen R.J.
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      The hasABC genes encode proteins required for synthesis of the antiphagocytic hyaluronic capsule.
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      Molecular analysis of the capsule gene region of group A Streptococcus: the hasAB genes are sufficient for capsule expression.
      All serotype M28 GAS strains have a naturally occurring nucleotide deletion in hasA that results in a nonfunctional HasA protein and loss of hyaluronic capsule production.
      • Bernard P.E.
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      • Olsen R.J.
      RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
      RocA is hypothesized to heterodimerize with CovS to affect CovR phosphorylation and downstream regulatory activity.
      • Horstmann N.
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      • Brumlow C.
      • DebRoy S.
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      • Makthal N.
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      Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus.
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      Regulatory rewiring confers serotype-specific hyper-virulence in the human pathogen group A Streptococcus.
      • Zhu L.
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      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      • Jain I.
      • Miller E.W.
      • Danger J.L.
      • Pflughoeft K.J.
      • Sumby P.
      RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus.
      However, a physical interaction between RocA and CovS (or any other transcription regulator) has not been definitively shown. RocA inactivation alters CovR phosphorylation and transcription of CovRS regulated genes.
      • Horstmann N.
      • Tran C.N.
      • Brumlow C.
      • DebRoy S.
      • Yao H.
      • Nogueras Gonzalez G.
      • Makthal N.
      • Kumaraswami M.
      • Shelburne S.A.
      Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus.
      • Miller E.W.
      • Danger J.L.
      • Ramalinga A.B.
      • Horstmann N.
      • Shelburne S.A.
      • Sumby P.
      Regulatory rewiring confers serotype-specific hyper-virulence in the human pathogen group A Streptococcus.
      • Zhu L.
      • Olsen R.J.
      • Horstmann N.
      • Shelburne S.A.
      • Fan J.
      • Hu Y.
      • Musser J.M.
      Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
      However, the molecular mechanism of the interaction remains unknown. Accessory proteins are often membrane proteins.
      • Buelow D.R.
      • Raivio T.L.
      Three (and more) component regulatory systems: auxiliary regulators of bacterial histidine kinases.
      • Eguchi Y.
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      • Yamane M.
      • Demizu R.
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      • Okada A.
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      B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli.
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      GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus.
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      The auxiliary protein complex SaePQ activates the phosphatase activity of sensor kinase SaeS in the SaeRS two-component system of Staphylococcus aureus.
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      The Abi-domain protein Abx1 interacts with the CovS histidine kinase to control virulence gene expression in group B Streptococcus.
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      SpdC, a novel virulence factor, controls histidine kinase activity in Staphylococcus aureus.
      Structural models predict that RocA folds similarly to an HK because of the presence of a predicted membrane-spanning region and putative HK domain (Figure 1).
      • Biswas I.
      • Scott J.R.
      Identification of rocA, a positive regulator of covR expression in the group A streptococcus.
      • Kelley L.A.
      • Mezulis S.
      • Yates C.M.
      • Wass M.N.
      • Sternberg M.J.E.
      The Phyre2 web portal for protein modeling, prediction and analysis.
      • Omasits U.
      • Ahrens C.H.
      • Muller S.
      • Wollscheid B.
      Protter: interactive protein feature visualization and integration with experimental proteomic data.
      However, Paluscio

      Paluscio E: Adaptive mechanisms to niche remodeling in Streptococcus pyogenes (dissertation). [St. Louis (MO)]: Graduate School of Arts & Sciences. Washington University in St. Louis

      reported that RocA lacks HK activity. Consistent with the putative H-box being dispensable for RocA function, the RocA R258K strain (with an amino acid change occurring in the putative H-box) (Figure 1) retained a WT-like transcriptome and virulence phenotype (Figure 6).
      Jain et al
      • Jain I.
      • Miller E.W.
      • Danger J.L.
      • Pflughoeft K.J.
      • Sumby P.
      RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus.
      have suggested that the amino-terminal transmembrane domains, but not the carboxy-terminal domain, contribute to RocA regulatory function. The seven predicted transmembrane-spanning domains
      • Kelley L.A.
      • Mezulis S.
      • Yates C.M.
      • Wass M.N.
      • Sternberg M.J.E.
      The Phyre2 web portal for protein modeling, prediction and analysis.
      • Omasits U.
      • Ahrens C.H.
      • Muller S.
      • Wollscheid B.
      Protter: interactive protein feature visualization and integration with experimental proteomic data.
      may enable RocA to heterodimerize with CovS or other TCS sensor proteins. Consistent with the idea that protein-protein interactions are key to wild-type RocA activity, our naturally occurring and isogenic mutant strains with rocA polymorphisms occurring in the transmembrane domains have rocA deletion-like transcriptomes (Figures 1 and 6). Drawing additional attention to the transmembrane domain, the isogenic RocA P97L and G184W mutant strains generated unexpected results relative to the parental WT strain and isogenic ΔrocA deletion mutant strain (Figure 6). Of note, codon 184 is also polymorphic (G184E) (Supplemental Figure S2) and function altering (Supplemental Figure S4) in serotype M89 GAS strains.
      • Beres S.B.
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      Transcriptome remodeling contributes to epidemic disease caused by the human pathogen Streptococcus pyogenes.
      In contrast to published studies,
      • Jain I.
      • Miller E.W.
      • Danger J.L.
      • Pflughoeft K.J.
      • Sumby P.
      RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus.
      we also demonstrated that the carboxy-terminal domain is essential to wild-type RocA activity in serotype M28 GAS (Figure 6). Mutations in codon 442, located in the putative nonfunctional HK ATPase domain (Figure 1), independently arose multiple times (T442P and T442I) and were propagated by vertical inheritance (n = 9 and 3 strains, respectively) (Figure 1). The isogenic T442P mutant strain had a rocA deletion-like transcriptome, virulence factor activity profile, and virulence phenotype in mice and NHPs (Figure 6). The repeated occurrence of polymorphisms at codon 442 strongly suggests selection for variation at this site in invasive strains. Additional studies of RocA are needed to fully elucidate the functional domains of RocA.
      In summary, we used transcriptome analysis, isogenic mutant strains, in vitro virulence factor assays, and mouse and NHP pathogenesis studies to investigate the effect of RocA amino acid variation on serotype M28 GAS biology, including pathogen-host interaction. Our study has brought novel insight into the role of an accessory protein in bacterial virulence and molecular pathogenesis.

      Acknowledgments

      We thank Samantha Kubiak and Hoang Nguyen for technical assistance, and Sasha M. Pejerrey for assistance in preparing the manuscript.
      R.J.O. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.

      Supplemental Data

      • Supplemental Figure S1

        Clustering of clinical isolates with naturally occurring rocA polymorphisms is growth phase dependent. Three-dimensional principal component analysis (PCA) of the RNA-sequencing data generated with the clinical isolates with naturally occurring rocA polymorphisms, an isogenic rocA deletion mutant (ΔrocA) strain, and four phylogenetically matched wild-type (WT) strains at midexponential (ME; A) and early-stationary (ES; B) growth phases. Each growth phase is represented by two PCA plots that are rotated approximately 180 degrees about the PC3 axis (ME) or the PC1 axis (ES). Clinical isolates are colored by clusters of the opposite growth phase, as determined by average-linkage hierarchical clustering. No obvious clustering is observed with this scheme.

      • Supplemental Figure S2

        rocA polymorphisms identified by population-based, whole genome sequencing studies of serotype M1, M12, M28, M59, and M89 GAS strains. The affected codon and amino acid change conferred by each polymorphism are shown. For polymorphisms due to nucleotide deletion or insertion, the affected nucleotide is identified. Alleles identified in multiple isolates are indicated. Polymorphisms that result in RocA protein truncation or presumed loss of rocA mRNA translation

        • Zhu L.
        • Olsen R.J.
        • Horstmann N.
        • Shelburne S.A.
        • Fan J.
        • Hu Y.
        • Musser J.M.
        Intergenic variable-number tandem-repeat polymorphism upstream of rocA alters toxin production and enhances virulence in Streptococcus pyogenes.
        are shown below the protein schematic, and polymorphisms that result in amino acid changes are shown above the protein schematic. Missense polymorphisms in the amino-terminal transmembrane domains are red. Predicted domains of the RocA protein are indicated.
        • Kelley L.A.
        • Mezulis S.
        • Yates C.M.
        • Wass M.N.
        • Sternberg M.J.E.
        The Phyre2 web portal for protein modeling, prediction and analysis.
        Predicted functional domains of the putative histidine kinase domain (H box, N box, F box, and G box) are identified.
        • Biswas I.
        • Scott J.R.
        Identification of rocA, a positive regulator of covR expression in the group A streptococcus.
        Asterisks denote stop codons. Daggers indicate strains that have two polymorphisms in rocA. Adapted from Bernard et al.
        • Bernard P.E.
        • Kachroo P.
        • Zhu L.
        • Beres S.B.
        • Eraso J.M.
        • Kajani Z.
        • Long S.W.
        • Musser J.M.
        • Olsen R.J.
        RocA has serotype-specific gene regulatory and pathogenesis activities in serotype M28 group A streptococcus.
        Copyright © American Society for Microbiology. HATPase, histidine kinase ATPase domain; MGE, mobile genetic element; TM, transmembrane domain.

      • Supplemental Figure S3

        Polymorphisms in RocA do not alter secreted streptococcal pyrogenic exotoxin B (SpeB) protease activity in a casein hydrolysis (milk plate) assay. P = 0.9710 (one-way analysis of variance with Dunn multiple comparisons test). Data are expressed as means ± SD. VNTR, variable number tandem repeat; WT, wild type.

      • Supplemental Figure S4

        The RocA G184E polymorphism in serotype M89 GAS results in decreased secreted NAD+-glycohydrolase (SPN) activity in vitro. Data are expressed as means ± SD. ****P < 0.0001 (one-way analysis of variance with Dunnett multiple comparisons test). MGAS11027, serotype M89 parental wild-type strain; MGAS26987, serotype M89 RocA G184E mutant; MGAS26989, serotype M89 RocA G184E and control of virulence sensor (CovS) R444I mutant; MGAS27003, serotype M89 RocA G184E and CovS F31I/E50G mutant; MGAS27010, serotype M89 RocA G184E mutant with an additional mobile genetic element disrupting the rocA gene.

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