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Microbial Immunity and Vaccines

Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity

Claudia M. Müller, Laura Conejero, Natasha Spink, Matthew E. Wand, Gregory J. Bancroft, Richard W. Titball
A. Camilli, Editor
Claudia M. Müller
aCollege of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, Devon, United Kingdom
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Laura Conejero
bLondon School of Hygiene and Tropical Medicine, London, United Kingdom
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Natasha Spink
bLondon School of Hygiene and Tropical Medicine, London, United Kingdom
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Matthew E. Wand
aCollege of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, Devon, United Kingdom
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Gregory J. Bancroft
bLondon School of Hygiene and Tropical Medicine, London, United Kingdom
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Richard W. Titball
aCollege of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, Devon, United Kingdom
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A. Camilli
Roles: Editor
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DOI: 10.1128/IAI.00178-12
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  • Fig 1
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    Fig 1

    Genetic organization and enzymatic activities of RelA-SpoT family proteins in B. pseudomallei and cell morphology of RelA-SpoT family mutants. (A) Schematic overview of the chromosomal regions surrounding the relA gene (BPSL1946 [top]) and the spoT gene (BPSL2561 [bottom]). Predicted promoters and their linear discriminant function (LDF) score are indicated. (B) Schematic of the classical synthesis pathway of (p)ppGpp. pppGpp is produced from GTP and ATP by either RelA- or SpoT-dependent mechanisms and is subsequently converted to ppGpp. ppGpp interacts directly with the RNA polymerase enzyme (RNAP) and thus affects gene expression. The effects of ppGpp induction are relieved by SpoT-mediated hydrolysis of ppGpp. (C to E) Cell morphology of B. pseudomallei wild-type strain K96243 (C), a K96243 ΔrelA single mutant (D), and a K96243 ΔrelA ΔspoT double mutant (E). All strains were grown to early stationary growth phase (24-h incubation at 37°C) in LB broth, and bacterial cells were visualized by bright-field microscopy at a magnification of ×100. The scale bars represent the average length of K96243 wild-type cells. In panel E, two images of K96243 ΔrelA ΔspoT mutant cells are presented, one focused on single cells (left) and one focused on a filament (right).

  • Fig 2
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    Fig 2

    Growth and stationary-phase survival of wild-type K96243 and isogenic mutants. All strains were inoculated into LB broth at an optical density of 0.01, and cultures were incubated at 37°C with aeration. (A) Growth curve representing the optical densities of the cultures at the indicated time points. (B) Stationary-phase survival of the strains determined by plating samples onto LB agar plates at the indicated time points. All experiments were performed in at least three independent experiments, and data are plotted on a logarithmic scale as means with standard deviations (A) or standard errors of the means (B) for those experiments.

  • Fig 3
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    Fig 3

    Intracellular survival of wild-type K96243 and isogenic mutants. J774A.1 murine macrophages were infected with wild-type K96243 and isogenic mutants at an MOI of 10, and intracellular bacterial numbers were determined at the indicated time points after initial infection by plating onto LB agar plates. Bars and error bars represent the means and standard deviations of a representative experiment performed in three technical replicates for each time point.

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    Fig 4

    Killing of G. mellonella larvae by wild-type K96243 and isogenic mutants. (A) Groups of 10 larvae per strain were injected with 1 × 104 CFU of bacteria and maintained at 37°C. Dead and live larvae were scored at indicated time points and plotted as Kaplan-Meier survival curves. All curves were significantly different from each other (log rank test; P < 0.01 in all cases). (B) The number of bacteria present inside the larvae at 20 h postinfection was determined by aseptically removing the bottom 2 mm of five larvae per strain, draining the hemocoel, and plating serial dilutions onto LB agar plates. Results are means and standard deviations from two independent experiments.

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    Fig 5

    Survival and protection of mice infected with wild-type K96243 and its isogenic ΔrelA ΔspoT mutant. (A and B) C57BL/6 mice were challenged intranasally with an intended dose of 2,500 CFU (acute challenge [A]) or 500 CFU (chronic challenge [B]). Actual infection doses for each experiment are given in parentheses. Log rank tests confirmed a significant difference in survival between the wild type and the ΔrelA ΔspoT mutant in both cases (P < 0.001 and P < 0.05, respectively). (C to E) C57BL/6 mice were immunized intranasally with 1 × 105 CFU of strain 2D2 or K96243 ΔrelA ΔspoT. After 5 weeks of incubation, mice were challenged intranasally with 1,000 CFU of B. pseudomallei strain 576. Mice were monitored for survival, and numbers are plotted as Kaplan-Meier survival curves (C). Log rank tests confirmed a significant difference in survival of the K96243 ΔrelA ΔspoT and the 2D2 cohorts compared to the saline control (P < 0.01 and P < 0.05, respectively). At day 55 after infection with the wild type, bacterial burdens were assessed in lungs (D) and spleens (E) of survivors. Note that only one mouse had survived up to this time point in the saline-treated sample group. The spleen of this mouse and that of one survivor in the K96243 ΔrelA ΔspoT sample group had extremely large abscesses and could not be harvested. N.A., not available.

Tables

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  • Table 1

    Strains and plasmids used in this study

    Strain or plasmidDescriptionReference
    B. pseudomallei strains
        K96243Clinical isolate from Thailand23
        K96243 ΔrelAInactivation of BPSL1946 (relA) by complete deletionThis study
        K96243 ΔrelA ΔspoTInactivation of BPSL1946 (relA) and BPSL2561 (spoT) by complete deletionThis study
        576Clinical isolate from Thailand2
        2D2Auxotroph transposon mutant of B. pseudomallei 5763
    E. coli strains
        DH5 ΔpirrecA1 gyrA (Nal) Δ(lacIZYA-argF) (ϕ80dlacΔ[lacZ]M15) pirRK643
        S17-1 ΔpirRPA-2 tra regulon; pirRK6 Smr Tpr43
    Plasmids
        pDM4Suicide vector with R6K origin; Cmr29
        pDM4-ΔrelA600 bp up- and downstream of relA cloned into pDM4 via XhoI/SphI sitesThis study
        pDM4-ΔspoT600 bp up- and downstream of spoT cloned into pDM4 via SalI/XmaI sitesThis study
  • Table 2

    Oligonucleotides used in this study

    NameSequence (5′–3′)aPurpose
    relA-1-fwCCCCTCGAGGAAGGCGCGCGGACAAGCGGMutagenesis
    relA-2-rvCGGCGCTCACTGCAGCATCGTCGGGTATGGCCAGTTMutagenesis
    relA-3-fwCCGACGATGCTGCAGTGAGCGCCGCCGGTGGGCCGCMutagenesis
    relA-4-rvCCCGCATGCCGTATCGACGAGTTCGCCGTCMutagenesis
    spoT-1-fwGCGGTCGACCTGCCGCCGTCGCTCGCGGCMutagenesis
    spoT-2-rvGCGGCGCTAACTAGTCATTTTCGCCTCCTGGGTTCGMutagenesis
    spoT-3-fwGCGAAAATGACTAGTTAGCGCCGCGCGGCGCCCGACMutagenesis
    spoT-4-rvGCGCCCGGGGCCTGCGCGAAATCGAGGTCGMutagenesis
    relA-up-fwACAACGGCACCGTCTATCTCConfirmation of plasmids
    relA-down-rvGCGGTGTCATAACCGTATTCConfirmation of plasmids
    spoT-up-fwCTCGCATTACCGTTGAAGACConfirmation of plasmids
    spoT-down-rvGCCCTTTCAGTTCGAAGTTGConfirmation of plasmids
    relA-3-fwGCGTGCAGTCGGACCTGCTGConfirmation of mutation
    relA-4-rvGTTCGGGAAAATTCGCAAGCCConfirmation of mutation
    relA-5-wtGTGGAAGCGGAATCGGCTGConfirmation of mutation
    spoT-3-fwGGTGAAGAAGCAGTTCCGCAConfirmation of mutation
    spoT-4-rvCGCCCGAAACAAATGGCGGConfirmation of mutation
    spoT-5-wtGCGGACGATGGTGTAGTGCConfirmation of mutation
    relA-RT-1AGATCCGCACGCAGGAAATGRT-PCR analysis
    relA-RT-2TTCGCTGTGCAGGTGGTAAGRT-PCR analysis
    spoT-RT-1GCGCATCAATGGCTCAAGTCRT-PCR analysis
    spoT-RT-2TTCAGGCGCATCGTCTTCAGRT-PCR analysis
    16S-RT-1GCCAGTCACCAATGCAGTTCRT-PCR normalization
    16S-RT-2ACCAAGGCGACGATCAGTAGRT-PCR normalization
    • ↵a Restriction sites are underlined. Complementary regions are in italics. Translational start and stop codons are in bold.

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Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity
Claudia M. Müller, Laura Conejero, Natasha Spink, Matthew E. Wand, Gregory J. Bancroft, Richard W. Titball
Infection and Immunity Aug 2012, 80 (9) 3247-3255; DOI: 10.1128/IAI.00178-12

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Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity
Claudia M. Müller, Laura Conejero, Natasha Spink, Matthew E. Wand, Gregory J. Bancroft, Richard W. Titball
Infection and Immunity Aug 2012, 80 (9) 3247-3255; DOI: 10.1128/IAI.00178-12
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