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Molecular Pathogenesis

Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Control of Carbohydrate Transport, Biofilm Formation, and Colonization of the Germfree Mouse Intestine

Laetitia Houot, Sarah Chang, Cedric Absalon, Paula I. Watnick
Laetitia Houot
Division of Infectious Disease, Children's Hospital Boston, Boston, Massachusetts
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Sarah Chang
Division of Infectious Disease, Children's Hospital Boston, Boston, Massachusetts
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Cedric Absalon
Division of Infectious Disease, Children's Hospital Boston, Boston, Massachusetts
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Paula I. Watnick
Division of Infectious Disease, Children's Hospital Boston, Boston, Massachusetts
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  • For correspondence: paula.watnick@childrens.harvard.edu
DOI: 10.1128/IAI.01356-09
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  • FIG. 1.
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    FIG. 1.

    Sugar specificities of PTS components as determined by an agar plate-based sugar fermentation assay. Wild-type (WT) V. cholerae and different mutants were assayed on MM agar plates containing a pH indicator and supplemented with N-acetylglucosamine (NAG) or other carbon sources as indicated. Medium acidification upon sugar fermentation leads to a yellow color. The strain key for agar plates is included below. The key is color coded as follows: blue, strains carrying mutations in EI homologs; red, strains carrying mutations in HPr homologs; and green, strains carrying mutations in EIIA homologs.

  • FIG. 2.
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    FIG. 2.

    PTS sugars activate PTS gene transcription. EI, EIIAGlc, HPr, and FPr transcript levels in wild-type V. cholerae grown in MM alone or MM supplemented with the indicated carbon sources were analyzed by quantitative RT-PCR. Three experimental replicates were performed. The data were analyzed using the ΔΔCT method for comparison to measurements from bacteria grown in MM alone. clpX was used as a standard.

  • FIG. 3.
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    FIG. 3.

    HPr and FPr repress biofilm-associated growth and vps gene transcription. The total growth and biofilm-associated growth of and vps transcription in wild-type (WT) V. cholerae and various PTS mutants were quantified. (A) Biofilm-associated and total growth in MM supplemented with glucose. (B) Biofilm-associated and total growth in MM supplemented with pyruvate. (C) β-Galactosidase activities of strains carrying a chromosomal vps-lacZ fusion at the lacZ site in MM supplemented with pyruvate. Error bars indicate the standard deviations of results from at least three experimental replicates. Measurements for the indicated V. cholerae mutants were compared to those for wild-type V. cholerae by using the t test of statistical significance. Biofilm measurements that are significantly different from wild-type biofilm measurements are marked with an asterisk (P < 0.0005). β-Galactosidase measurements for all mutants were significantly different from that for the wild-type strain (P < 0.01). Furthermore, the β-galactosidase activity of the ΔEI mutant was significantly different from that of the ΔHPr ΔFPr mutant (P = 0.002).

  • FIG. 4.
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    FIG. 4.

    HPr and FPr must be phosphorylated to repress biofilm-associated growth. (A) Quantification of total and biofilm-associated growth of wild-type (WT) V. cholerae harboring a pBAD expression vector carrying a control sequence (pCTL) or of a ΔHPr ΔFPr mutant harboring a pBAD expression vector carrying either a control sequence (pCTL), the wild-type HPr gene (pHPr), a sequence encoding an unphosphorylatable form of HPr (pHPr{H15A}), the wild-type FPr gene (pFPr), a sequence encoding an unphosphorylatable form of FPr (FPr{H324A}), or a sequence encoding the C-terminal HPr-like domain of FPr including residues 309 to 401 (pFPrtrunc) in MM supplemented with pyruvate. Protein expression was induced with 0.04% l-arabinose. Schematic representations of the rescue constructs provided in trans are illustrated above the data. Error bars indicate the standard deviations of results from three experimental replicates. Asterisks indicate measurements significantly different from the measurement for the ΔHPr ΔFPr (pCTL) strain (P < 0.0014). (B) Western blots demonstrating expression of the relevant protein from the rescue plasmid. A V5 epitope tag was used for visualization.

  • FIG. 5.
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    FIG. 5.

    HPr and FPr are downstream of EI in the pathways regulating biofilm growth. (A) Quantification of total and biofilm-associated growth of a ΔPTS mutant and a ΔPTS ΔFPr mutant harboring a pBAD expression vector carrying either a control sequence (pCTL) or the wild-type gene encoding EI (pEI) in MM supplemented with pyruvate. Protein expression was induced with 0.04% l-arabinose. Error bars indicate the standard deviations of results from three experimental replicates. The asterisk indicates a measurement significantly different from that for the ΔPTS (pCTL) mutant (P = 0.0019). (B) Western blot demonstrating expression of the relevant protein from the rescue plasmid. A V5 epitope tag was used for visualization. WT, wild type.

  • FIG. 6.
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    FIG. 6.

    V. cholerae persists in the distal portion of the adult germfree mouse intestine for 1 month. (A) Enumeration of V. cholerae CFU in stool pellets over time. Mice were infected by having free access to V. cholerae-inoculated saline solution for 24 h. At each indicated time point, two stool pellets were collected from each mouse. Stool pellets from cohoused mice were pooled. The samples were weighed and homogenized in 1 ml of PBS. Serial dilutions of this suspension were spread onto LB agar plates, and the resulting colonies were enumerated. Data were normalized with respect to stool weight. This experiment included 10 mice housed 2 to a cage. Cage 1 mice were monitored for 6 days, mice in cages 2 and 3 were monitored for 13 days, and cage 4 and 5 mice were monitored for 23 days. (B) Quantification of V. cholerae bacteria in the proximal small intestine (1), the middle small intestine (2), the distal small intestine (3), the cecum (4), and the large intestine (5) at 6 and 36 days postinoculation. Measurements at 6 days are shown in blue, while measurements at 36 days are shown in red. Bars represent the geometric means of measurements. Numbers of CFU in all segments of the small intestine were statistically significantly different from those in the large intestine (P = 0.0286). (C) Micrographs of different hematoxylin- and eosin-stained portions of the small intestine of a V. cholerae-infected, previously germfree mouse. The intestine was fixed in neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Bar, 10 μm.

  • FIG. 7.
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    FIG. 7.

    V. cholerae is found primarily in the lumen of the large intestine rather than on the epithelial surface. Micrographs of Gram-stained sections of the small intestine of a previously germfree, V. cholerae-infected mouse are shown. Bacteria are indicated by arrows. Bar, 10 μm.

  • FIG. 8.
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    FIG. 8.

    ΔEI and ΔEI Δvps mutants have a competitive disadvantage in the germfree mouse intestine. Exponentially grown wild-type (WT) V. cholerae bacteria were mixed at an approximately 1:1 ratio with either ΔEI mutant bacteria (A) or ΔEI ΔvpsL mutant bacteria (B). The resulting Vibrio suspensions were diluted 20-fold in saline and used to inoculate four mice housed in two cages. On the indicated days, fecal pellets were collected and plated onto LB agar to determine total numbers of CFU. Samples were also plated onto sucrose pH indicator agar to determine the mutant/WT ratio. The WT strain, which is able to ferment sucrose, forms large yellow colonies on this medium, and the ΔEI and ΔEI ΔvpsL mutants, which are unable to transport sucrose, form small green colonies.

Tables

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  • TABLE 1.

    Bacterial strains and plasmids

    Strain or plasmidGenotype or descriptionSource or reference
    E. coli SM10λpir thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu (λpirR6K) Kmr 21
    V. cholerae strains
        PW357MO10 lacZ::vpsLp→lacZ Smr 8
        PW751MO10 lacZ::vpsLp→lacZ ΔPTS Smr 12
        PW836MO10 lacZ::vpsLp→lacZ ΔEIIAGlc Smr 12
        PW961MO10 lacZ::vpsLp→lacZ ΔEI Smr 12
        PW964MO10 lacZ::vpsLp→lacZ ΔHPr Smr 12
    MO10 lacZ::vpsLp→lacZ ΔFPr SmrThis study
    MO10 lacZ::vpsLp→lacZ ΔNPr SmrThis study
        PW965MO10 lacZ::vpsLp→lacZ ΔHPr ΔFPr SmrThis study
    MO10 lacZ::vpsLp→lacZ ΔHPr ΔNPr SmrThis study
    MO10 lacZ::vpsLp→lacZ ΔPTS ΔFPr SmrThis study
    MO10 lacZ::vpsLp→lacZ ΔEIIANtr1 SmrThis study
    MO10 lacZ::vpsLp→lacZ ΔEIIANtr2 SmrThis study
    MO10 lacZ::vpsLp→lacZ VC0672::pGP704 Smr AprThis study
    MO10 lacZ::vpsLp→lacZ VCA1045::pGP704 Smr AprThis study
    MO10 lacZ::vpsLp→lacZ VCA0245::pGP704 Smr AprThis study
    MO10 lacZ::vpsLp→lacZ VC1820::pGP704 Smr AprThis study
    MO10 lacZ::vpsLp→lacZ VC1822::pGP704 Smr AprThis study
    MO10 lacZ::vpsLp→lacZ VC1826::pGP704 Smr AprThis study
        PW249MO10This study
    MO10 ΔEI SmrThis study
    MO10 ΔEI ΔvpsL SmrThis study
    Plasmids for construction of deletions
        pWM91oriR6K mobRP4 lacI pTac tnp mini-Tn10(Km) Kmr Apr 20
        pWM91ΔPTSpWM91 carrying an unmarked, in-frame deletion of the PTS operon 12
        pWM91ΔEIIAGlcpWM91 carrying an unmarked, in-frame deletion in VC0964 12
        pWM91ΔEIpWM91 carrying an unmarked, in-frame deletion in VC0965 12
        pWM91ΔHPrpWM91 carrying an unmarked, in-frame deletion in VC0966 12
        pWM91ΔNPrpWM91 carrying an unmarked, in-frame deletion in VC2533This study
        pWM91ΔFPrpWM91 carrying an unmarked, in-frame deletion in VCA0518This study
        pWM91ΔEIIANtr1pWM91 carrying an unmarked, in-frame deletion in VC2531This study
        pWM91ΔEIIANtr2pWM91 carrying an unmarked, in-frame deletion in VC1824This study
    Plasmids for construction of insertions
        pGP704::VC1820pGP704 carrying an internal fragment of VC1820This study
        pGP704::VC1822pGP704 carrying an internal fragment of VC1822This study
        pGP704::VC1826pGP704 carrying an internal fragment of VC1826This study
        pGP704::VCA1045pGP704 carrying an internal fragment of VCA1045This study
        pGP704::VCA0245pGP704 carrying an internal fragment of VCA0245This study
        pGP704::VC0672pGP704 carrying an internal fragment of VC0672This study
        pGP704::VC1283pGP704 carrying an internal fragment of VC1283This study
    Plasmids used in rescue experiments
        pBAD-TOPO-EIpBAD-TOPO carrying the coding sequence of VC0965 12
        pBAD-TOPO-HPrpBAD-TOPO carrying the coding sequence of VC0966 12
        pBAD-TOPO-HPr(H15A)pBAD-TOPO carrying a VC0966 variant encoding an H-to-A mutation at position 15This study
        pBAD-TOPO-FPrpBAD-TOPO carrying the coding sequence of VCA0518This study
        pBAD-TOPO-FPr(H324A)pBAD-TOPO carrying a VCA0518 variant encoding an H-to-A mutation at position 324This study
        pBAD-TOPO-FPr(309-401)pBAD-TOPO carrying a fragment of VCA0518 encoding positions 309 to 401This study
  • TABLE 2.

    Primers used in this study

    Primer functionRelevant gene or gene productPrimer nameSequence
    Gene deletionFPrLH55TGCTCCACCACAGCCATCACT
    LH56TAACGAGCGGCCGCACATTCTTAACTCCTGTCTGCC
    LH57TGCGGCCGCTCGTTAGGCTTAGGCGAAGGTTAAGG
    LH58TCATGCTGCGCAGTTGGGCA
    NPrLH43CTGCTTGGCCCACTCAAAG
    LH44TAACGAGCGGCCGCACATAGGGGCTCCTAGGATTG
    LH45TGCGGCCGCTCGTTAGAGTAAAGCTCCTCACTAGC
    LH46GTGACACATCATCCGGCAAG
    EIIANtr1LH121CTCAGTGACAGCAAGATCGCA
    LH122TAACGAGCGGCCGCAGGTGCAGTCCAATGAAAGTAC
    LH123TGCGGCCGCTCGTTAAGCGATCAAGAGCTGTACAACATC
    LH124GACTGAAACACCATCACAAGGTC
    EIIANtr2LH49TCGCTAGAGTAACGAGCGAT
    LH50TAACGAGCGGCCGCACATCATGCCAAGGATCAGCG
    LH51TGCGGCCGCTCGTTACTGATGTACGCGATGTCACGC
    LH52TGTTGCTGCAAGCTCTCCTTCA
    Gene insertionVC1820LH87ATATGAATTCCTCATCAGTTAAATACTTTATGAGCC
    LH88TATAGAATTCTTACATAAATATGGTGGTCTGCGCC
    VC1822LH89ATATGAATTCCCCATCTGATCGAACCTGAAATC
    LH90TATAGAATTCTTACGGCTGGTAATCGGCATAATCCAT
    VC1826LH91ATATGAATTCGCAAGCGAATTCCAAACAAGCCGT
    LH92TATAGAATTCTTAATCAGCCCTTTGCTTTCTGCTTGG
    EINtrLH71ATATGAATTCTTGCGCGCATGTCGGATGTTTATC
    LH72TATAGAATTCTTATCGATTCGTGCGCCATCAAGAGTA
    VC1283LH192ATATGAATTCGATTCAGTTGGGCTTTGTCGGTA
    LH193TATAGAATTCTTACCTGTGCCAGAATTGTCGTCA
    VCA0245LH151ATATGAATTCCAACTCAATCCAACTGCAAGCCAAAGCC
    LH152TATAGAATTCTTACTACGCGATGTGCGCTAATTCTTGTTGG
    VCA1045LH105ATATGAATTCGGAGAACATTCACCTTGGCCTGAA
    Mutant rescueFPrLH61TTAGAACTCACTACACAAGATATTC
    LH62ACCTTCGCCTAAGCCAGCATTGATC
    FPr(309-401)aLH195CGCGCTCATACGGCGACTTT
    HPr(H15A)LH17AAAACGGCCTTGCCACTCGT
    LH18ACGAGTGGCAAGGCCGTTTT
    FPr(H324A)LH59AAATAGCCATGGTCTGGCCGCTCGTC
    LH60GACGAGCGGCCAGACCATGGCTATTT
    Use in quantitative RT-PCREIACCGTTATCGAAGAGCAAGCCACT
    TCTGCGCAGTTTCAGAAGGCGTTA
    EIIAGlcGCGATCAAGCCTGCTGGTAACAAA
    AAGCCTTCACCTTTCAGCTCAACC
    HPrTGTTCAAGCTGCAAACGCTAGGTC
    GCAACTAGGTGCTCAACAGCTTCT
    FPrATCACTGAGGAAACGATAGCCGCA
    ACTTGACCATCGCCATCCAGGTTA
    ClpXAGAGTTCATTGGTCGTCTGCCTGT
    AACAACGCCGCATACTGTTTGGTC
    • ↵ a FPr(309-401), fragment of FPr comprising positions 309 to 401.

  • TABLE 3.

    Summary of PTS component transport specificities

    ComponentGeneE. coli orthologSpecificitya for:
    GlucoseNAGSucroseTrehaloseMaltoseFructoseMannitolMannose
    EIVC0965EIXXXXXX
    VC0672EINtr
    HPrVC0966HPrXXXXXX
    VCA0518FPrX
    VC2533NPr
    EIIAVC0964EIIAGlcXXX
    VC1283
    VC1820
    VC1822
    VC1824EIIANtr2
    VC1826X
    VC2531EIIANtr1
    VCA0245
    VCA0518FPrX
    VCA1045X
    • ↵ a X denotes specificity for the indicated sugar. NAG, N-acetylglucosamine.

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Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Control of Carbohydrate Transport, Biofilm Formation, and Colonization of the Germfree Mouse Intestine
Laetitia Houot, Sarah Chang, Cedric Absalon, Paula I. Watnick
Infection and Immunity Mar 2010, 78 (4) 1482-1494; DOI: 10.1128/IAI.01356-09

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Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Control of Carbohydrate Transport, Biofilm Formation, and Colonization of the Germfree Mouse Intestine
Laetitia Houot, Sarah Chang, Cedric Absalon, Paula I. Watnick
Infection and Immunity Mar 2010, 78 (4) 1482-1494; DOI: 10.1128/IAI.01356-09
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KEYWORDS

biofilms
Carbohydrate Metabolism
Intestines
Phosphoenolpyruvate Sugar Phosphotransferase System
Vibrio cholerae

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