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Research Article | Host-Microbe Biology

Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate

Bailey M. Carignan, Kyle D. Brumfield, Mike S. Son
Michael J. Imperiale, Editor
Bailey M. Carignan
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Kyle D. Brumfield
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Mike S. Son
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Michael J. Imperiale
University of Michigan
Roles: Editor
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DOI: 10.1128/mSphere.00253-16
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  • FIG 1
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    FIG 1

    CT production profiles of single-SNP strains and double-, triple-, and quadruple-SNP combination strains. SNPs and SNP combinations were introduced into the WT strain N16961, and average fold changes in CT production levels relative to the CT production in WT N16961 were calculated. Other controls included MQ1795 (hypervirulent clinical isolate) and O395ΔtoxT (negative control). Strains containing the different SNPs and SNP combinations introduced into WT N16961 are indicated by the numbers beginning with “MS” found in Table 2. The presence of SNPs identified in genes in the indicated clinical isolates is denoted by a “+,” whereas the WT N16961 version of a SNP is denoted by a “−.” A two-tailed Student t test yielded P values of ≤0.05 (*), <0.005 (**), and <0.0005 (***).

  • FIG 2
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    FIG 2

    TcpA production profiles of single-SNP strains and double-, triple-, and quadruple-SNP combination strains. WCE were prepared, 3 µg of total protein was loaded onto a 16% Tris-glycine polyacrylamide gel, and proteins in immunoblots were quantified using densitometry for V. cholerae virulence factor TcpA. Immunoblots (inset) are from different blots (indicated by borders), all with WT N16961 and other controls run simultaneously. Nonspecific bands serve as a loading control (L.C.) and are labeled accordingly. Average fold changes were calculated relative to the TcpA level in N16961. Other controls included MQ1795 (hypervirulent clinical isolate), classical O395, El Tor C6706, O395ΔtoxT, and O395ΔtcpA. Strains containing the different SNPs and SNP combinations introduced into WT N16961 are indicated by the numbers beginning with “MS” found in Table 2. The presence of SNPs identified in genes in the indicated clinical isolates is denoted by a “+,” whereas the WT N16961 version of a SNP is denoted by a “−.” A two-tailed Student t test yielded P values of ≤0.05 (*), <0.005 (**), and <0.0005 (***).

  • FIG 3
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    FIG 3

    ToxT production profiles of single-SNP strains and double-, triple-, and quadruple-SNP combination strains. WCE were prepared, 12 µg of total protein was loaded onto a 14% Tris-glycine polyacrylamide gel, and proteins in immunoblots were quantified using densitometry for V. cholerae virulence factor ToxT. Immunoblots (inset) are from different blots (indicated by borders), all with WT N16961 and other controls run simultaneously. Nonspecific bands serve as a loading control (L.C.) and are labeled accordingly. Average fold changes were calculated relative to the ToxT level in N16961. Other controls included MQ1795 (hypervirulent clinical isolate), classical O395, El Tor C6706, O395ΔtoxT, and O395ΔtcpA. Strains containing the different SNPs and SNP combinations introduced into WT N16961 are indicated by the numbers beginning with “MS” found in Table 2. The presence of SNPs identified in genes in the indicated clinical isolates is denoted by a “+,” whereas the WT N16961 version of a SNP is denoted by a “−.” A two-tailed Student t test yielded P values of ≤0.05 (*), <0.005 (**), and <0.0005 (***).

  • FIG 4
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    FIG 4

    Schematic representation of the involvement of each of the four genes on virulence factor expression. HapR binds to the aphA promoter, resulting in a decrease in virulence factor production, and H-NS decreases virulence factor production through a mechanism of transcriptional silencing. LuxO works in a manner contrary to that of HapR, and VieA degrades c-di-GMP, both of which result in indirect upregulation of virulence factor production. qrr, quorum regulatory RNA; sRNA, small RNA.

Tables

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  • TABLE 1

    SNPs previously identified in MQ1795 by next-generation deep sequencinga

    GeneGene identifierBase no.N16961MQ1795
    BaseResidueBaseResidue
    hapR VC0583219None (T deletion)NonfunctioningT (insertion)Functioning
    hns VC1130319GGAS
    luxO VC1021656CATV
    vieA VC1652235CLTF
    • ↵ a Single nucleotide polymorphisms (SNPs) of interest found in the hypervirulent MQ1795 strain compared to the El Tor reference strain N16961. MQ1795 has a gain of function in the hapR gene with a mutation that results in the insertion of a thymine. The remaining SNPs (hns, luxO, and vieA) result in neither a gain of function nor a loss of function but rather result in residue changes.

  • TABLE 2

    Escherichia coli and Vibrio cholerae strains used in this studya

    StrainDescriptionReference or source
    Escherichia coli
    S17-λpir recA thi pro hsdR− M+ [RP4-2-Tc::Mu::Kmr Tn7] (λpir) Tmpr Strrde Lorenzo and Timmis (49)
    Vibrio cholerae
        O395Classical wild type, StrrSon et al. (9)
        C6706El Tor wild type, StrrSon et al. (9)
        N16961El Tor reference strain, StrrSon et al. (9)
        MQ1795El Tor variant, clinical isolateNair et al. (27)
        MS0005Classical O395ΔtoxT, StrSon et al. (9)
        MS0014Classical O395ΔtcpA, StrKirn et al. (50)
        MS0235El Tor N16961 hapR* StrThis study
        MS0245El Tor N16961 hns* StrThis study
        MS0230El Tor N16961 luxO* StrThis study
        MS0240El Tor N16961 vieA* StrThis study
        MS0256El Tor N16961 hapR* hns* StrThis study
        MS0254El Tor N16961 hapR* luxO* StrThis study
        MS0251El Tor N16961 hapR* vieA* StrThis study
        MS0285El Tor N16961 hns* luxO* StrThis study
        MS0281El Tor N16961 hns* vieA* StrThis study
        MS0287El Tor N16961 luxO* vieA* StrThis study
        MS0279El Tor N16961 hapR* hns* luxO* StrThis study
        MS0295El Tor N16961 hapR* hns* vieA* StrThis study
        MS0297El Tor N16961 hapR* luxO* vieA* StrThis study
        MS0299El Tor N16961 hns* luxO* vieA* StrThis study
        MS0282El Tor N16961 hapR* hns* luxO* vieA* StrThis study
    • ↵ a S17-λpir was used to introduce all possible SNPs and SNP combinations into the El Tor reference strain N16961, using a previously described allelic-exchange protocol (46). SNPs introduced are indicated by asterisks. Tmpr represents trimethoprim resistance, and Strr represents streptomycin resistance.

  • TABLE 3

    Primers used in this study to introduce SNPs and verify sequencing

    Oligonucleotide no.Oligonucleotide nameSequence (5′–3′)aPurpose
    0159ETV-hapR-ForwardGATCGGAATTCCCATTTCCTACTTGAAGCTGTAGCGGTGTTGGCAGAllelic exchange
    0160ETV-hapR-SapI-ReverseGATCGGCTCTTCACGTCAGTACTCCAACTTCTTGACCGATCACATCGAllelic exchange
    0161ETV-hapR-SapI-ForwardGATCGGCTCTTCAACGAACCACAAAATTCAGCACATCGTCAACCAAGTCTTCAllelic exchange
    0162ETV-hapR-ReverseGATCGAGATCTCAGTATCGCTGACTTTGGTGGCGCGTATAGTACCAllelic exchange
    0163ETV-luxO-ForwardGATCGGAATTCGCTGGATATTGATATCAATATCGTGGGTACCGGAllelic exchange
    0164ETV-luxO-SapI-ReverseGATCGGCTCTTCACGCCTTGACGCTCAGTCGCCGCCCCAGTAAAAGCAllelic exchange
    0165ETV-luxO-SapI-ForwardGATCGGCTCTTCAGCGTGGCAGAAGCGGCTGATGGGGGAACCCTCTTTTTGGAllelic exchange
    0166ETV-luxO-ReverseGATCGAGATCTGCTTGTTCAATGGCTTGTTTTTCGGTCATCCACAGCAllelic exchange
    0167ETV-hns-ForwardGATCGGAATTCGGTTTATTTATGGCGGGTTACACCGAAGATTCCGAllelic exchange
    0168ETV-hns-SapI-ReverseGATCGGCTCTTCAGTGAAGAAAAAACTTGGACAGGCCAAGGCCGTACTCCAllelic exchange
    0169ETV-hns-SapI-ForwardGATCGGCTCTTCACACTGTTGGTGTCGATATACTTGTATTTTGCAGGGCGAGGAllelic exchange
    0170ETV-hns-ReverseGATCGAGATCTCAATCATTTCATTGATACTTATTTCATAAAAACACCAllelic exchange
    0171ETV-vieA-ForwardGATCGGAATTCGGTCGGGGTTCCGTTGTACACCTCATGCTCCGTCAllelic exchange
    0172ETV-vieA-SapI-ReverseGATCGGCTCTTCATCAGCGCTGTGGAAGATACGATTCTTGAGTTAACAllelic exchange
    0173ETV-vieA-SapI-ForwardGATCGGCTCTTCATGAATATCACCACACCTAGCTTAGGTGCCTGTAAACTCAGAllelic exchange
    0174ETV-vieA-ReverseGATCGAGATCTCAGTCGTTATCTCACAACACAATGGGTCGCACTGCCAllelic exchange
    0212 hapR-UpstreamGCATTGTATAAATGGGGCTTGGAGAATTTAGGCGSequencing
    0213 hapR-DownstreamGCTCAGTGATCTGTTGACCTAATTCGCGAATGCGSequencing
    0214 hns-UpstreamGCATTAGCTTTAACAGGAGAAAGCGATCCGCTCGCSequencing
    0215 hns-DownstreamGCAACTAGGTTCCAGTGAGAAAAACAAGTGCCACAGCSequencing
    0216 luxO-UpstreamGGCTAGGCTATGCAACATAATCAATCTTTGCAGSequencing
    0217 luxO-DownstreamCCAAGTTTGCAGCTTGCGATAGATGGTTGACGGSequencing
    0218 vieA-UpstreamGCAGTTGAGCCAGTTGACCTAATGACGCGTAACCSequencing
    0219 vieA-DownstreamGCGGAACAAGCTCTGCAAGCGGGTATGGATAAGGSequencing
    • ↵ a Italics represent regions of enzyme restriction sites.

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Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate
Bailey M. Carignan, Kyle D. Brumfield, Mike S. Son
mSphere Sep 2016, 1 (5) e00253-16; DOI: 10.1128/mSphere.00253-16

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Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate
Bailey M. Carignan, Kyle D. Brumfield, Mike S. Son
mSphere Sep 2016, 1 (5) e00253-16; DOI: 10.1128/mSphere.00253-16
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    • ABSTRACT
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KEYWORDS

Toxin-coregulated pilus
Vibrio cholerae
cholera toxin
hapR
hns
luxO
toxT
vieA
virulence factors

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