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Molecular Pathogenesis

Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization

Jutta Nesper, Stefan Schild, Crystal M. Lauriano, Anita Kraiss, Karl E. Klose, Joachim Reidl
Jutta Nesper
1Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany
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Stefan Schild
1Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany
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Crystal M. Lauriano
2University of Texas Health Science Center, San Antonio, Texas 78229-3900
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Anita Kraiss
1Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany
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Karl E. Klose
2University of Texas Health Science Center, San Antonio, Texas 78229-3900
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Joachim Reidl
1Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany
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  • For correspondence: joachim.reidl@mail.uni-wuerzburg.de
DOI: 10.1128/IAI.70.11.5990-5996.2002
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  • FIG. 1.
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    FIG. 1.

    Surface polysaccharides expressed by LPS and CPS mutants. (A) Western blot analysis with O139-specific antiserum (see Materials and Methods). Reactive bands are indicated as follows: the upper band represents the capsular form of O139 side chain, and the lower band corresponds to the O side chain/core OS/lipid A portion. (B) Silver-stained polyacrylamide gel after SDS-PAGE. Stained bands are indicated as follows: the upper bands represent the O side chain/core OS/lipid A, and the lower bands are the core OS/lipid A portion. Lanes: wt, MO10 wild type; 1, MO10 ΔwaaL; 2, MO10 ΔwaaL pBADwaaL; 3, MO10 ΔgalEK; 4, MO10 ΔgalEK pACYCgalE; 5, MO10 wbfF; 6, MO10 wavB; 7, MO10 wavB pSSkanwavB2; 8, MO10 waaF; 9, MO10 waaF pACYCwaaF; 10, MO10 ΔwaaL ΔwbfF.

  • FIG. 2.
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    FIG. 2.

    Colonization deficiency of LPS and CPS mutants. Each point represents the value from a single mouse competition assay. The competitive index is defined as the output ratio of mutant to wild-type bacteria divided by the input ratio of mutant to wild-type bacteria. Strains with the wild-type ability to colonize have a competitive index of approximately 1.0. Data sets: 1, MO10 wbfF; 2, MO10 ΔgalEK; 3, MO10 ΔgalEK pACYCgalE; 4, MO10 ΔwaaL; 5, MO10 ΔwaaL pBADwaaL; 6, MO10 wavB; 7, MO10 wavB, pSSkanwavB2; 8, MO10 waaF; 9, MO10 waaF, pACYCwaaF; 10, MO10 ΔwaaL ΔwbfF. The defect in colonization of all mutant strains was statistically significant (P < 0.01) compared with the wild-type strain MO10 by Student's two-tailed t test.

  • FIG. 3.
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    FIG. 3.

    Expression of TcpA and cholera toxin in LPS and CPS mutants. Strains were grown under inducing conditions (Materials and Methods). (A) Whole-cell lysates were subjected to Western blot with TcpA antiserum. (B) Cell-free supernatants were measured for cholera toxin (CT) by ELISA and given in per optical density unit at 600 nm. Lanes: 1, MO10 (noninduced); 2, MO10 (induced); 3, MO10 wbfF (induced); 4 MO10 ΔwaaL (induced); 5, MO10 ΔgalEK (induced); 6, MO10 wavB (induced); 7, MO10 waaF (induced); 8, MO10 waaF pACYCwaaF (induced).

  • FIG. 4.
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    FIG. 4.

    Growth phenotypes of LPS and core OS mutants on TCBS plates. (A) TCBS plus ampicillin (100 μg/ml) containing 0.8% bile. (B) TCS plus ampicillin (100 μg/ml) without bile. (C) TCBS containing 0.8% bile. Sets: 1, MO10 pBAD28; 2, MO10 waaF pACYCwaaF; 3, MO10 waaF; 4, MO10 wavB pSSkanwavB2; 5, MO10 wavB; 6, MO10 ΔgalEK; 7, MO10 ΔwaaL; 8, MO10 wbfF.

Tables

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  • TABLE 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidRelevant genotypeaReference or source
    Strains
        MO10Clinical isolate of V. cholerae O139 42
        PW96MO10 lacZ::MCS 43
        MO10 ΔwaaLMO10 ΔwaaLThis study
        MO10 ΔwaaLwbfFMO10 ΔwaaL wbfF::pGP704This study
        MO10 ΔgalEKΔgalE::cm ΔgalKThis study
        MO10 wbfF wbfF::pGP704This study
        MO10 waaF waaF::pGP704This study
        MO10 wavB wavB::pGP704This study
    Plasmids
        pTrcAkanCloning vector Kmr 24
        pBAD28Cloning vector Apr Kanr 8
        pGP704OriR6K mobRP4 Apr 22
        pUC4KApr Kmr 39
        pGPwaaF‘waaF’b Apr 25
        pGPwavB‘wavB’ Apr 25
        pKEK229OriR6K mobRP4 sacB Apr 6
        pKEKΔgalE::cmΔgalE::cm Apr 26
        pBADwaaL waaL + Kmr 25
        pACYCgalE galE + Kmr 26
        pACYCwaaF waaF + Kmr 25
        pAKwavB wavB tetR + Cmr 25
        pGPwbfF‘wbfF’ in pGP704 AprThis study
        pKEKΔgalKΔgalK in pKEK229 AprThis study
        pKEKΔwaaLΔwaaL in pKEK229 AprThis study
        pSSkanwavB1pAKwavB tetR+ Cmr KmrThis study
        pSSkanwavB2pSSkanwavB1 ΔtetR Cmr KmrThis study
    • ↵ a MCS, multiple cloning site.

    • ↵ b ‘gene,’ internal gene fragment.

  • TABLE 2.

    Resistance to antimicrobial peptides and the complement system

    StrainMBC of protamine (μg/ml)Serum resistancea (% survival)
    MO1015096.75 ± 6.50
    MO10 ΔwaaL15099.15 ± 1.69
    MO10 wbfF1503.68 ± 5.58
    MO10 ΔwaaL ΔwbfF1500.03 ± 0.03
    MO10 ΔgalEK1500.05 ± 0.06
    MO10 ΔgalEK pACYCgalEND90.25 ± 11.84
    MO10 waaFb75100 ± 0
    MO10 waaF pACYCwaaF150ND
    MO10 wavB15092.48 ± 8.68
    • ↵ a Serum resistance was calculated as the ratio of the number of CFU found after cells were incubated for 45 min in 20% normal human serum divided by the number of CFU found after cells were incubated for 45 min in 20% heat-inactivated normal human serum. Indicated is the mean ± standard deviation. All assays were performed at least four times. ND, not determined.

    • ↵ b This mutant was also tested in the presence of control plasmid pTrcAkan.

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Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
Jutta Nesper, Stefan Schild, Crystal M. Lauriano, Anita Kraiss, Karl E. Klose, Joachim Reidl
Infection and Immunity Nov 2002, 70 (11) 5990-5996; DOI: 10.1128/IAI.70.11.5990-5996.2002

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Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
Jutta Nesper, Stefan Schild, Crystal M. Lauriano, Anita Kraiss, Karl E. Klose, Joachim Reidl
Infection and Immunity Nov 2002, 70 (11) 5990-5996; DOI: 10.1128/IAI.70.11.5990-5996.2002
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KEYWORDS

Bacterial Capsules
Intestines
Lipopolysaccharides
Vibrio cholerae

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