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Editor's Pick Research Article | Host-Microbe Biology

Capacity To Utilize Raffinose Dictates Pneumococcal Disease Phenotype

Vikrant Minhas, Richard M. Harvey, Lauren J. McAllister, Torsten Seemann, Anna E. Syme, Sarah L. Baines, James C. Paton, Claudia Trappetti
Larry S. McDaniel, Editor
Vikrant Minhas
aResearch Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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Richard M. Harvey
aResearch Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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Lauren J. McAllister
aResearch Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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Torsten Seemann
bMelbourne Bioinformatics, The University of Melbourne, Melbourne, Australia
cDepartment of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia
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Anna E. Syme
bMelbourne Bioinformatics, The University of Melbourne, Melbourne, Australia
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Sarah L. Baines
cDepartment of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia
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James C. Paton
aResearch Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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Claudia Trappetti
aResearch Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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Larry S. McDaniel
University of Mississippi Medical Center
Roles: Editor
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Jeffrey Weiser
NYU Lagone Health
Roles: Solicited external reviewer
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Andrew Camilli
Tufts University School of Medicine
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DOI: 10.1128/mBio.02596-18
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Figures

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  • FIG 1
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    FIG 1

    Differential growth of blood and ear isolates in raffinose. S. pneumoniae serotype 14 ST15 blood isolates 4559 and 4534 and ear isolates 947 and 51742 were grown in 200 µl CDM supplemented with 0.5% glucose (CDM+Glc) or 0.5% raffinose (CDM+Raf). Similar growth studies were also performed for serotype 3 ST180 strains 180/15 (blood isolate) and 180/2 (ear isolate). OD600 was measured every hour for 12 h. Data are mean OD600 ± standard deviation (SD) from triplicate assays.

  • FIG 2
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    FIG 2

    Genetic loci encoding raffinose uptake and utilization in S. pneumoniae. The numbers below each gene refer to the locus tags in the serotype 14 ST15 947 genome. The locations of SNPs in serotype 14 ST15 and serotype 3 ST180 isolates are indicated with asterisks; horizontal arrows show the locations of promoters.

  • FIG 3
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    FIG 3

    Expression of raffinose pathway genes by serotype 14 and 3 blood and ear isolates. The indicated strains were grown in CDM+Glc to an OD600 of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR using 16S rRNA as an internal control (see Materials and Methods). The data presented are the means ± SD from three independent experiments. *, P < 0.05, **, P < 0.01, and ****, P < 0.0001, by unpaired t test.

  • FIG 4
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    FIG 4

    Growth phenotype and raffinose pathway gene expression in serotype 23F ST81 blood and ear isolates. (A) Growth of blood isolate 5076 and ear isolate 9725241 in CDM+Glc or CDM+Raf was monitored by OD600 for 12 h. Data are mean OD600 ± SD from triplicate assays. (B) The indicated strains were grown in CDM+Glc to an OD600 of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR using 16S rRNA as an internal control. The data presented are the means ± SD from three independent experiments. ***, P < 0.001 by unpaired t test.

  • FIG 5
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    FIG 5

    Growth phenotype and raffinose operon gene expression in rafR exchange mutants. (A) S. pneumoniae strains 4559, 947, 4559947rafR, and 9474559rafR were grown in CDM+Glc or CDM+Raf, and OD600 was monitored for 12 h. Data are mean OD600 ± SD from triplicate assays. (B) The indicated strains were grown in CDM+Glc to an OD600 of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR. Data are the means ± SD from three independent experiments. *, P < 0.05, ***, P < 0.001, and ****, P < 0.0001, by unpaired t test.

  • FIG 6
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    FIG 6

    Virulence phenotype of rafR exchange mutants. Groups of 16 mice were infected intranasally with 108 CFU of the indicated strain. At 24 h, all mice from each group were euthanized and numbers of pneumococci in the indicated tissues/sites were quantitated (see Materials and Methods). Viable counts (total CFU per tissue) are shown for each mouse at each site; horizontal bars indicate the geometric mean (GM) CFU for each group; the broken line indicates the threshold for detection. Differences in GM bacterial loads between groups are indicated by asterisks: *, P < 0.05, **, P < 0.01, and ****, P < 0.0001, by unpaired t test.

  • FIG 7
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    FIG 7

    Growth phenotype and raffinose operon gene expression in ΔrafK mutants. (A) S. pneumoniae serotype 3 strains 180/15, 180/2, 180/15ΔrafK, and 180/2ΔrafK were grown in CDM+Glc or CDM+Raf, and OD600 was monitored for 12 h. Data are mean OD600 ± SD from triplicate assays. (B) The indicated strains were grown in CDM+Glc to an OD600 of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR. Data are the means ± SD from three independent experiments. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, by unpaired t test.

Tables

  • Figures
  • TABLE 1

    Genes containing indels or SNPs that led to amino acid changes, identified from the whole-genome variant calling analysis between 4559 and 947a

    Locus tag
    in 947
    GeneProductChange in aa sequence in 4559 relative to 947
    0862pfkAATP-dependent 6-phosphofructokinaseS212G
    1153glgAGlycogen synthaseE174G
    1345pncBNicotinate phosphoribosyltransferaseN434D
    1631scrRHTH-type transcriptional regulatorΔL27-G28
    1803rafRHTH-type transcriptional regulatorD249G
    1255pyrPUracil permeaseV65A
    1737piuAFe3+ import ATP-binding proteinG141V
    2020ABC transporter ATP-binding proteinY508N
    0330cpsECPS glycosyltransferaseL43P
    1139igaImmunoglobulin A1 proteasePremature stop 1905 (4559) due to indel
    1594nanBSialidase BPremature stop 362 (947) due to indel
    1741pfbAPlasmin and fibronectin-binding protein AT318M
    0945coiACompetence proteinE78K
    1141addAATP-dependent helicase/nuclease subunit AI980M
    1060Acetyl transferaseC101G
    1194Cytosolic protein containing multiple CBS domainsPremature stop 104 (947) due to SNP
    1731Hypothetical protein (no Pfam match)H32P
    • ↵a Results for the raffinose pathway gene rafR are in boldface.

  • TABLE 2

    Genes containing indels or SNPs that led to amino acid changes, identified from the whole-genome variant calling analysis between 180/2 and 180/15a

    Locus tag
    in 180/2
    GeneProductbChange in aa sequence of 180/15 relative to 180/2
    100purNPhosphoribosyl-glycinamideG81A
    254rpsJ30S ribosomal protein S10Y58D
    285Hypothetical proteinA81S
    314cdsAPhosphatidate cytidylyltransferaseM14I
    335adhPAlcohol dehydrogenase 1M210V
    403fabKEnoyl-[acyl-carrier-protein] reductaseI1029T
    449Nitronate monooxygenaseI55M
    512glnAGlutamine synthetaseF22L
    645nhaKSodium, potassium, lithium and rubidium/H+ antiporterG190D
    741VanZ family proteinC149W
    996Hypothetical proteinH38R
    1121clcAH+/Cl− exchange transporterM131I
    1138ptsHPhosphocarrier protein HPrI14V
    1172Formate/nitrate transporterA211E
    1194glnPGlutamine transport system permease proteinS662A
    1234SpF43_sRNAY31C
    1306alaSAlanine-tRNA ligaseE18A
    1387apbEFAD:protein FMN transferaseM52I
    1404LPXTG cell wall anchor domain-containing proteinΔK112-Q119; G125K, E126T, P127E, E130V,
    K131N, I133D; ΔQ135-P178
    1491rafKRaffinose import ATP-binding proteinI227T
    1616dnaBDNA helicaseC375R
    1760fepD_2Ferric enterobactin transport system permease proteinS248G
    1863rpoCDNA-directed RNA polymerase subunit betaD76E
    1878acyPAcylphosphataseV4I
    1887rsgASmall ribosomal subunit biogenesisG40S
    2045aspSAspartate tRNA ligaseE51V
    2100dltDd-Alanyl-lipoteichoic acid biosynthesis proteinD151E
    • ↵a Results for the raffinose pathway gene rafK are in boldface.

    • ↵b FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.

  • TABLE 3

    S. pneumoniae strains used in this study

    StrainDescriptionSourceReference
    4559Serotype 14 ST15Blood7
    947Serotype 14 ST15Ear7
    4534Serotype 14 ST15Blood7
    51742Serotype 14 ST15Ear7
    4559947rafR4559 expressing 947 rafR geneThis study
    9474559rafR947 expressing 4559 rafR geneThis study
    180/15Serotype 3 ST180Blood6
    180/2Serotype 3 ST180Ear6
    5076Serotype 23F ST81BloodThis study
    9725241Serotype 23F ST81EarThis study
  • TABLE 4

    Oligonucleotide primers used in this study

    PrimerSequence (5′→3′)Reference
    rafR Flank FGCGAACGTAGGTTACAATCGTThis study
    rafR R j tailGGAAAGGGGCCCAGGTCTCTCTAGCATGTGCTACCTCCTACCThis study
    rafR F j tailCATTATCCATTAAAAATCAAAGGGGAAATCCTACCAAGCTGTCTACCThis study
    rafR Flank RCGAACGTAGTTCAGTGGTAGAAThis study
    Janus FCCGTTTGATTTTTAATGGATAATG33
    Janus RAGAGACCTGGGCCCCTTTCC33
    aga FAAGGTCAGAATGGTCCACAGThis study
    aga RGCTGGAAAATCAGCCATAAAThis study
    rafG FCCTATGGCAGCCTACTCCATCThis study
    rafG RGGGTCTGTGGAATCGCATAGGThis study
    rafK FAACGACGTAGCTCCAAAAGAThis study
    rafK RGCTGGTTTACGTTCCAAGAAThis study
    16s rRNA FGGTGAGTAACGCGTAGGTAA34
    16s rRNA RACGATCCGAAAACCTTCTTC34
    rafR sangerAGTAGAAGAGCTGGTGTTTGThis study
    rafR sangerTCTGTGACTAAGCCAGTTTCThis study
    rafK Flank FAGGACTTGGTTCTTGTTGAGThis study
    rafK R ery tailTTGTTCATGTAATCACTCCTTCCTACCATGAGGTGAACTCCThis study
    rafK F ery tailCGGGAGGAAATAATTCTATGAGATCAGTTAATCTAGGGAGAGThis study
    rafK Flank RCTCAAAGGCAACTGGACAACThis study
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Capacity To Utilize Raffinose Dictates Pneumococcal Disease Phenotype
Vikrant Minhas, Richard M. Harvey, Lauren J. McAllister, Torsten Seemann, Anna E. Syme, Sarah L. Baines, James C. Paton, Claudia Trappetti
mBio Jan 2019, 10 (1) e02596-18; DOI: 10.1128/mBio.02596-18

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Capacity To Utilize Raffinose Dictates Pneumococcal Disease Phenotype
Vikrant Minhas, Richard M. Harvey, Lauren J. McAllister, Torsten Seemann, Anna E. Syme, Sarah L. Baines, James C. Paton, Claudia Trappetti
mBio Jan 2019, 10 (1) e02596-18; DOI: 10.1128/mBio.02596-18
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    • ABSTRACT
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KEYWORDS

Streptococcus pneumoniae
carbohydrate metabolism
otitis media
pneumonia
single nucleotide polymorphisms
virulence

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