Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Molecular Pathogenesis

Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA

Rita Tamayo, Stefan Schild, Jason T. Pratt, Andrew Camilli
Rita Tamayo
Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stefan Schild
Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jason T. Pratt
Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrew Camilli
Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: andrew.camilli@tufts.edu
DOI: 10.1128/IAI.01337-07
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    cdpA encodes a c-di-GMP PDE. (A) The domain structure of the VC0130 protein, CdpA, is shown. CdpA is a three-domain protein containing an N-terminal domain of unknown function (COG3287), a GGDEF, and an EAL domain. The divergent amino acid sequences present in cdpA at the GG(D/E)EF and EAL motifs are indicated in the relevant domains. The amino acids bounding each domain are noted below. (B) Lysates from AC1817 [VieA], AC1835 [VieA(E170A)], AC2376 [CdpA], and AC2642 [CdpA(AVGAW)] were assayed for protein expression by Western blot analysis. The WT and respective mutant proteins were comparably expressed. No His6-tagged protein was detected in vector-only controls (not shown). (C) Lysates containing the indicated proteins as well as buffer- and vector-only controls were tested for the ability to hydrolyze radiolabeled c-di-GMP. The reactions were analyzed by TLC; products and corresponding Rf values are indicated on the right.

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    The degenerate GGDEF domain of CdpA is required for optimal PDE activity of the EAL domain. Altered PDE activity of the mutated proteins was determined based on their effects on in vitro c-di-GMP-regulated phenotypes in the cdpA strain, namely, motility and biofilm production. (A) The mean diameters of motility are given in mm for strains containing empty vector, pCdpA, pCdpAΔEAL, or pAVGAW (CdpA containing a mutated GGDEF motif). The medium contained 50 μM IPTG to induce gene expression and Km to maintain the plasmids. (B) The amount of biofilm formed by the above-described strains grown under static conditions in the presence of 50 μM IPTG is shown. In both figures, asterisks indicate significant changes from WT. The pCdpA and pAVGAW strains had significantly different motility (P = 9 × 10−5) and biofilm formation (P = 6 × 10−4) levels.

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    CdpA regulates a subset of c-di-GMP-regulated processes. (A) CdpA negatively regulates biofilm production. Shown are the mean A570 values reflecting crystal violet staining of the biofilms formed by WT, the cdpA strain, and the complemented cdpA(pCdpA) strain. The amount of IPTG present in the medium is indicated above each sample. All strains showed significant differences in biofilm formation from WT by Student's t test (P < 0.05). (B) CdpA does not regulate the motility of V. cholerae. Motility was assessed in soft agar medium and is presented as the mean diameter of motility (mm). (C) CdpA is dispensable for V. cholerae colonization of the infant mouse. The cdpA strain was competed against the fully virulent lacZ mutant strain AC66; the cdpA(pCdpA) complementation strain was competed against AC2365 containing empty vector. Each shape represents a CI from a single animal. The horizontal bars indicate the median CI.

  • FIG. 4.
    • Open in new tab
    • Download powerpoint
    FIG. 4.

    Expression of cdpA during infection of the mouse. Expression of cdpA during infection of the mouse was measured at 7 h and 21 h p.i. using RIVET. Expression in shaking broth culture (37°C) was included as a control. Expression of ctxA, a virulence gene known to be induced early during infection, was analyzed in parallel. Transcription of cdpA was not induced at 7 h p.i. compared to what was seen in vitro but was activated by 21 h p.i. (P = 0.002). In contrast, transcription of ctxA was induced by an early time point during infection (P = 0.001). In both figures, each symbol represents the percentage of CFU that were Km sensitive (resolved) under each condition at each time point in an individual sample. Horizontal lines indicate the median percent resolved CFU.

  • FIG. 5.
    • Open in new tab
    • Download powerpoint
    FIG. 5.

    Ectopic modulation of intracellular c-di-GMP by expression of vdcA. (A) To directly test the ability of vdcA expression to increase intracellular c-di-GMP, 2D-TLC was done to detect intracellular c-di-GMP in strains containing pVdcA (AC1902), pVdcAi (AC2389), or vector alone (AC1903). The c-di-GMP and GMP spots are indicated. (B) The pVdcA, pVdcAi, and vector-only strains were tested for the ability to activate biofilm formation. The mean intensities of crystal violet staining of the biofilms (A570) are shown. (C) The pVdcA, pVdcAi, and vector strains were tested for decreased motility. The mean diameters of motility in mm from three independent assays are shown. In panels B and C, asterisks indicate significant differences in biofilm and motility, respectively, compared to WT as determined by Student's t test.

  • FIG. 6.
    • Open in new tab
    • Download powerpoint
    FIG. 6.

    The effects of increased c-di-GMP on virulence of V. cholerae. (A) The pVdcA and pVdcAi strains were competed against WT containing vector alone in mice. In addition, the VdcA and VdcA(I) strains were competed directly to control for any effects of protein levels. Each symbol represents the CI obtained from an individual mouse. The horizontal bars indicate the geometric mean CI. Asterisks denote statistically significant differences in colonization (P < 1 × 10−4). (B) Transcription of toxT during infection of the mouse in the presence of WT (pVdcAi) and ectopically increased c-di-GMP (pVdcA) was measured using RIVET. The percentage of CFU that were Tc sensitive, and therefore the percentage of bacteria that have expressed toxT, at the indicated time points p.i. are shown. Each data point represents the mean from at least three mice. Asterisks indicate significant differences in resolution rate at that time point as determined by Student's t test.

Tables

  • Figures
  • TABLE 1.

    Strains used in this study

    StrainDescriptionReference or source
    V. cholerae strains
        AC51El Tor C6709, Smr 39
        AC66C6709 lacZ::res-tet-res 7
        AC1901C6709 pMMB67EH::vieA-His6This work
        AC1902C6709 pMMB67EH::VCA0956(pVdcA)This work
        AC1903C6709 lacZ::res-tet-res pMMB67EHThis work
        AC2131C6709 cdpA::pGP704This work
        AC2280C6709 ΔvpsRThis work
        AC2284C6709 ΔvpsR pMMB67EH::vieA-His6This work
        AC2286C6709 ΔvpsR pVdcAThis work
        AC2365C6709 lacZ::res-tet-res pMMBneoThis work
        AC2377C6709 cdpA::pGP704 pMMBneo::cdpA-His6This work
        AC2380C6709 pMMBneo::cdpA-His6This work
        AC2389C6709 pMMB67EH::VCA0956(E258A) pVdcA(I)This work
        AC2390C6709 pMMB67EH::vieA(E170A)This work
        AC2395C6709 lacZ pVdcAThis work
        AC2434C6709 cdpA::pGP704 pMMBneoThis work
        AC2435C6709 ΔvpsR pMMBneoThis work
        AC2483C6709 toxT::tnpR lacZ::res-tet-res pVdcAThis work
        AC2484C6709 toxT::tnpR lacZ::res-tet-res pVdcA(I)This work
        AC2595C6709 lacZ::res-neo-sacB-resThis work
        AC2606C6709 cdpA::tnpR lacZ::res-sacB-neo-resThis work
        AC2655C6709 pMMBneo::cdpA(AVGAW)-His6This work
        AC2658C6709 cdpA::pGP704 pMMBneo::cdpAΔEAL-His6This work
        AC2659C6709 cdpA::pGP704 pMMBneo::cdpA(AVGAW)-His6This work
    E. coli strains
        DH5αF− Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1Laboratory strain
        DH5αλPirF− Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1 λ::pirLaboratory strain
        SM10λPir thi recA thr leu tonA lacY supE RP4-2-Tc::Mu λ::pirLaboratory strain
        AC575DH5α pMMB67EH 33
        AC751DH5αλPir pCVD442lac 11
        AC2364DH5α pMMBneo 35
        AC1817DH5α pMMB67EH::vieA-His6 48
        AC1835DH5α pMMB67EH::vieA(E170A)-His6 48
        AC1887DH5α pVdcAThis work
        AC2129SM10λPir pGP704::′cdpA′aThis work
        AC2142SM10λPir pCVD442lac::ΔvpsRThis work
        AC2376DH5α pMMBneo::cdpA-His6This work
        AC2386DH5α pVdcAThis work
        AC2528SM10λPir pRes 35, 44
        AC2599SM10λPir pIVET5ncdpAThis work; 44
        AC2641DH5α pMMBneo::cdpAΔEAL-His6This work
        AC2642DH5α pMMBneo::cdpAAVGAW-His6This work
    • ↵ a An internal fragment of cdpA was cloned in order to make the plasmid insertion.

  • TABLE 2.

    Primers used in this study

    Primer nameSequencea (5′ to 3′) (reference)
    oriRCAGCAGTTCAACCTGTTG
    67EHFCGACATCATAACGGTTCTGG
    67EHRTTCACTTCTGAGTTCGGCAT
    0130FXGGTCTAGAAGAGCGGTTTGTATGCGATT
    0130RXGGTCTAGAGCCGGCTCAAACGAGTATAG
    0130PFTAATCGCCCTGAAAGTGACC
    C0130FTTCTAGATTTAGGATACATTTTTATGTTTACGGTCTCGC
    C0130RTTGCATGCCTAATGGTGATGGTGATGGTGACCCAAGCGTGAAGGT
    0130EALTTCTAGATTTAGGATACATTTTTATGGCGGTGGTACGTGATGAT
    vpsRF1TGCATGCCTACAACCCAAATCACGC
    vpsRR1AATCAGCAAAACTTACATGAACCTATATTCCTT
    vpsRF2GAATATAGGTTCATGTAAGTTTTGCTGATTTAC
    vpsRR2TTTCTAGAGGTAAACTCAAGCCGATT
    vpsRF0CTCTGTGGCGTTAGAAG
    vpsRR0CCTGTCCTTAGTGATGTG
    A0956F1CCGAGCTCTTTAGGATACATTTTTGTGATGACAACTGAAGAT
    A0956R1GGGCATGCAGTTTAGAGCGGCATGAC
    A0956EAFGGCGGTGAAGCGTTTGCACTG
    A0956EARCAGTGCAAACGCTTCACCGCC
    0130GEFTTCTAGATTTAGGATACATTTTTATGACGGACAATTTGGCAC
    0130GAF GCTGTCGGTGCTTGGGCAACGGTTTTT
    0130GARCCAAGCACCGACAGCAATCGCATACAAACC
    0130EAFTACGCTTGTTTAGTGCGGATTGAA
    0130EARCACTAAACAAGCGTAGGAAGCCAC
    0130CGRTTGCATGCCTAATGGTGATGGTGATGGTGCTCCTGACGAACACTTTC
    0130GASGCGATTGCTGTCGGTGCT
    0130EASATTGTGGCTTCCTACGCT
    3287qFTTCTCTCCTTTGAGTCGCGAGCAT
    3287qRATTTGCAGCAACGTCACCTGATGG
    vpsRqFTGGATTCAGTACCTGGCTCT
    vpsRqRCGCAATCCCGTTAAGGCTAA
    RPB2FDCTGTCTCAAGCCGGTTACAA (38)
    RPB2RVTTTCTACCAGTGCAGAGATGC (38)
    • ↵ a Restriction sites are underlined; sequence encoding the His6 tag is both underlined and in boldface; boldface text without underlining indicates codons changed to obtain desired amino acid mutations.

  • TABLE 3.

    c-di-GMP levels in cdpA strains

    StrainDescriptionRatio of c-di-GMP to GMPFold change in c-di-GMPa
    AC61WT0.054N/A
    AC2131 cdpA 0.1993.7
    AC2377 cdpA(pCdpA)0.1532.8
    AC2377 cdpA(pCdpA) + IPTG0.0571.1
    • ↵ a Data from one representative experiment are shown. N/A, not applicable.

  • TABLE 4.

    Induction of transcription in biofilm and during infectiona

    TranscriptComparisonFold changeb
    vpsR Biofilm/broth 24°C7.0 ± 1.4*
    cdpA Biofilm/broth 24°C3.9 ± 1.0*
    cdpA In vivo/broth 37°C8.1 ± 6.0**
    • ↵ a Cycle thresholds for cdpA and vpsR transcripts were normalized to those for rpoB in each sample.

    • ↵ b *, P = 0.03 by Mann-Whitney U test; **, P = 0.008 by Mann-Whitney U test.

PreviousNext
Back to top
Download PDF
Citation Tools
Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA
Rita Tamayo, Stefan Schild, Jason T. Pratt, Andrew Camilli
Infection and Immunity Mar 2008, 76 (4) 1617-1627; DOI: 10.1128/IAI.01337-07

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA
Rita Tamayo, Stefan Schild, Jason T. Pratt, Andrew Camilli
Infection and Immunity Mar 2008, 76 (4) 1617-1627; DOI: 10.1128/IAI.01337-07
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Bacterial Proteins
cholera
Cyclic GMP
Phosphoric Diester Hydrolases
Vibrio cholerae

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522