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Molecular Pathogenesis

Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli

Grant T. Donovan, J. Paul Norton, Jean M. Bower, Matthew A. Mulvey
S. M. Payne, Editor
Grant T. Donovan
Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, Utah, USA
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J. Paul Norton
Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, Utah, USA
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Jean M. Bower
Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, Utah, USA
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Matthew A. Mulvey
Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, Utah, USA
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S. M. Payne
Roles: Editor
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DOI: 10.1128/IAI.00796-12
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  • Fig 1
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    Fig 1

    Impaired use of alternative carbon sources by UTI89ΔcyaA and UTI89Δcrp. (A) MacConkey agar plate streaked with UTI89, UTI89ΔcyaA, and UTI89Δcrp. Growth of UTI89, UTI89ΔcyaA, UTI89Δcrp, and complemented mutants in modified M9 medium (B) and modified M9 medium lacking glucose (C). Mutant strains were complemented by uninduced expression of cyaA or crp from a Ptac promoter. Strains transformed with the empty vector pRR48 served as controls. Each curve reflects the means of results from a single experiment and is representative of at least three independent experiments performed in quadruplicate.

  • Fig 2
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    Fig 2

    UPEC requires functional cAMP-CRP for virulence in the murine urinary tract. Adult female CBA/J mice (A, B) or C3H/HeJ mice (C) were infected via catheterization with 1 × 107 CFU of wild-type UTI89 or isogenic mutants lacking cyaA or crp. Graphs show bacterial titers present in the bladder at 6 h (A) and 3 days (B, C) postinoculation. Bars indicate median values for each group; n ≥ 11 mice. P values were determined using Mann-Whitney U tests.

  • Fig 3
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    Fig 3

    UTI89ΔcyaA and UTI89Δcrp are sensitive to nitrosative stress and methyl viologen but resistant to H2O2. Growth of UTI89, UTI89ΔcyaA, and UTI89Δcrp in MES-LB broth (A), MES-LB broth + 1 mM ASN (B), MES-LB broth + 1 mM H2O2 (C), LB broth (D), and LB broth containing 1 mM MV (E). Growth curves show the means of results from a single experiment and are representative of at least three independent experiments carried out in quadruplicate.

  • Fig 4
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    Fig 4

    Complementation of UTI89ΔcyaA and UTI89Δcrp in the presence of nitrosative and oxidative stresses. Graphs show growth of UTI89 versus UTI89ΔcyaA (A to C) and UTI89Δcrp (D to F) in MES-LB (A, D), MES-LB ± 1 mM ASN (B, E), or MES-LB ± 1 mM H2O2 (C, F), all without added IPTG. Strains carried pcyaA, pcrp, or the control plasmid pRR48, as indicated. Growth curves show the means of results from a single experiment and are representative of at least three independent experiments carried out in quadruplicate.

  • Fig 5
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    Fig 5

    H2O2 resistance correlates with increased RpoS expression and catalase activity in UTI89ΔcyaA and UTI89Δcrp. (A) Western blot of RpoS (σS) in UTI89, UTI89ΔcyaA, and UTI89Δcrp after growth to mid-exponential phase in LB broth. Relative levels of RpoS normalized to loading control (Ctrl) are indicated. (B, C) Curves show growth of UTI89 and UTI89Δcrp carrying empty vector pRR48 or prpoS, as indicated, in MES-LB ± 1 mM H2O2. (D) Graph of catalase activity in UTI89, UTI89ΔcyaA, UTI89Δcrp, and UTI89/prpoS following growth to stationary phase in LB broth + 1 mM IPTG. Data are expressed relative to wild-type UTI89 as the means ± standard errors of three independent experiments carried out in triplicate. (E) Levels of trehalose present in UTI89ΔfimH, UTI89ΔotsBA, UTI89ΔcyaA, and UTI89Δcrp following growth to stationary phase (OD600 = 1.0). The ΔfimH mutant carries the same chloramphenicol resistance cassette as the ΔcyaA and Δcrp mutants and served as the control. (F, G) Graphs show growth of UTI89 and its mutant derivatives (ΔcyaA, Δcrp, and Δdps mutants) carrying pRR48 or pdps, as indicated, in MES-LB ± 1 mM H2O2. Each growth curve (B, C, F, and G) shows the means of results from a single experiment and is representative of at least three independent experiments carried out in quadruplicate. Dps and RpoS expression in these assays was induced by addition of 0.5 mM IPTG. (H) Survival of UTI89Δdps complemented with empty vector pRR48 or pdps following 15 min of exposure to 0.05 M CuSO4. Results were obtained without addition of IPTG and are presented relative to those of wild-type UTI89/pRR48. The indicated P values were determined using two-tailed unpaired t tests.

  • Fig 6
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    Fig 6

    Expression of RpoS or Dps enables wild-type UTI89 to survive low-pH stress at levels similar to those of the ΔcyaA and Δcrp mutants. After reaching mid-logarithmic growth phase in LB broth, UTI89 and the UTI89ΔcyaA, UTI89Δcrp, UTI89Δdps, UTI89ΔrpoS, UTI89ΔcrpΔdps, and UTI89ΔcrpΔrpoS mutants (± pRR48, pdps, and prpoS, as indicated) were exposed to acid (pH 3.5) stress for 30 min. Following washes in PBS, numbers of surviving bacteria were determined by dilution plating. Plasmid-containing strains were grown in the presence of 0.5 mM IPTG prior to challenge with pH stress. Data are expressed as the means ± standard deviations of three independent experiments. P values of <0.05 are indicated by one asterisk (*) for comparison with UTI89 and by two asterisks (**) for comparison with UTI89/pRR48, as determined by two-tailed unpaired t tests.

Tables

  • Figures
  • Table 1

    Bacterial strains and plasmids

    Strain or plasmidDescriptionSource
    Strains
        UTI89UPEC strain (cystitis isolate, O18:K1:H7)25
        UTI89ΔcyaAUTI89cyaA::Clmr (pKD3)This study
        UTI89ΔcrpUTI89crp::Clmr (pKD3)This study
        UTI89ΔdpsUTI89dps::Kanr (TT23691)This study
        UTI89ΔfimHUTI89fimH::Clmr (pKD3)83
        UTI89ΔotsBAUTI89otsBA::Kanr (pKD4)This study
        UTI89ΔrpoSUTI89rpoS::Kanr (TT23691)This study
        UTI89ΔcyaAΔotsBAUTI89cyaA::Clmr (pKD3) otsBA::Kanr (pDK4)This study
        UTI89ΔcrpΔrpoSUTI89crp::Clmr (pKD3) rpoS::Kanr (TT23691)This study
        UTI89ΔcrpΔdpsUTI89crp::Clmr (pKD3) dps::Kanr (TT23691)This study
        TT23691Strain with Kanr cassette flanked by universal primer sites84
    Plasmids
        pRR48Ampr cloning plasmid containing an IPTG-inducible Ptac promoter upstream of the MCS26
        pKM208Ampr plasmid; encodes IPTG-inducible lambda red recombinase27
        pKD3Template plasmid for gene disruption; contains FRTa-flanked Clmr cassette28
        pKD4Template plasmid for gene disruption; contains FRT-flanked Kanr cassette28
        pcrpAmpr plasmid; crp (from UTI89) cloned into PstI, KpnI sites of pRR48This study
        pcyaAAmpr plasmid; cyaA (from UTI89) cloned into PstI, KpnI sites of pRR48This study
        pdpsAmpr plasmid; IPTG-inducible expression of E. coli Dps55
        prpoSAmpr plasmid; rpoS (from UTI89) cloned into PstI, HindIII sites of pRR48This study
    • ↵a FRT, FLP recombination target.

  • Table 2

    Primer sequences

    PrimerSequence
    crp KOa
        ForwardGCGCATGGTGCTTGGCAAACCGCAAACAGACCCGACTCTCTGTGTAGGCTGGAGCTGCTTCG
        ReverseCGCGCTACCAGGTAACGCGCCACTCTGACGGGATTAACGACATATGAATATCCTCCTTAG
    crp KO confirmation
        ForwardGTATGCAAAGGACGCCACAT
        ReverseTTCGCCAAGCATTAACCCAA
    cyaA KO
        ForwardGCGGAATCACAGTCATGACGGGTAGCAAATCAGGCGATACTGTGTAGGCTGGAGCTGCTTCG
        ReverseTACTGCTGCAACAGCGGCGCGTCATGCTCCTGATTGGCAGCATATGAATATCCTCCTTAG
    cyaA KO confirmation
        ForwardAACCAGGCGCGAAAAGTGGT
        ReverseCTGAAAGGCGACGAGTGGAT
    otsBA KO
        ForwardATGTCTGTAAAGCGCGTTCTGCGCAACACAATAAGAAATGTGTAGGCTGGAGCTGCTTCG
        ReverseCTACGCAAGCTTAGGAAAGGTAGCAACTTTATCGCGCTGCCATATGAATATCCTCCTTAG
    otsBA KO confirmation
        ForwardAGCGAAACGCACTGTCTGAT
        ReverseTTGCCTACGGTGAGTTAAGC
    dps KO
        ForwardTTATTCGATGTTAGACTCGATAAACCACAGGAATTTATCCAGGTCGCGAGCACCAAACACCCCCCAAAACC
        ReverseGTGATAGGAACAGCCAGAATAGCGGAACACATAGCTGGTGCTATACTTAGCACACAACCACACCACACCAC
    dps KO confirmation
        ForwardGATAGCAGATGGATGCACTA
        ReverseTGACAGTACGCAAAGAGAGC
    rpoS KO
        ForwardCCAGCCTCGCTTGAGACTGGCCTTTCTGACAGATGCTTACCACCAAACACCCCCCAAAACC
        ReverseTGCCGCAGCGATAAATCGGCGGAACCAGGCTTTTGCTTGACACACAACCACACCACACCAC
    rpoS KO confirmation
        ForwardAATGATGATTGCCGAATGTGACGCTG
        ReverseGCATTGTGTCGTTATGGGCGTAGG
    pcrp
        ForwardCCCCC CTGCAG ATGGTGCTTGGCAAACCGCA
        ReverseCCCCC GGTACC TTAACGAGTGCCGTAAACGA
    pcyaA
        ForwardCCCCC CTGCAG TTGTACCTCTATATTGAGAC
        ReverseCCCCC GGTACC TCACGAAAAATACTGCTGCA
    prpoS
        ForwardCATTC CTGCAG ATGTTCCGTCAAGGGATCA
        ReverseAGTGC AAGCTT TTATTCGCGGAACAGCGCT
    pRR48 sequencing primer
        ForwardCTGCTGAAGAGTACTTTGG
        ReverseCCAAAGCTGAAGACATCCAG
    • ↵a KO, knockout.

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Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli
Grant T. Donovan, J. Paul Norton, Jean M. Bower, Matthew A. Mulvey
Infection and Immunity Dec 2012, 81 (1) 249-258; DOI: 10.1128/IAI.00796-12

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Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli
Grant T. Donovan, J. Paul Norton, Jean M. Bower, Matthew A. Mulvey
Infection and Immunity Dec 2012, 81 (1) 249-258; DOI: 10.1128/IAI.00796-12
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