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Molecular Pathogenesis

Hyperinduction of Host Beta Interferon by a Listeria monocytogenes Strain Naturally Overexpressing the Multidrug Efflux Pump MdrT

Kierstyn T. Schwartz, Joshua D. Carleton, Sarah J. Quillin, Stuart D. Rollins, Daniel A. Portnoy, Jess H. Leber
A. Camilli, Editor
Kierstyn T. Schwartz
aDepartment of Microbiology, The University of Chicago, Chicago, Illinois, USA
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Joshua D. Carleton
aDepartment of Microbiology, The University of Chicago, Chicago, Illinois, USA
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Sarah J. Quillin
aDepartment of Microbiology, The University of Chicago, Chicago, Illinois, USA
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Stuart D. Rollins
aDepartment of Microbiology, The University of Chicago, Chicago, Illinois, USA
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Daniel A. Portnoy
bDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA
cSchool of Public Health, University of California, Berkeley, Berkeley, California, USA
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Jess H. Leber
aDepartment of Microbiology, The University of Chicago, Chicago, Illinois, USA
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A. Camilli
Roles: Editor
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DOI: 10.1128/IAI.06286-11
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  • Fig 1
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    Fig 1

    L. monocytogenes strain LO28 hyperinduces the macrophage production of IFN-β during cytosolic infection. RT-qPCR analysis of IFN-β (A) and IL-1β (B) induction in macrophages infected with the indicated strains at 4 h postinfection, with MOIs of ∼0.5 to 3.0 (see Materials and Methods for the normalization procedure). *, statistical difference in induction compared to induction by strain 10403S using Student's t test (P ≤ 0.05); **, P ≤ 0.01. (C and D) Analysis of IFN-β induction, as in panel A, in WT and myd88−/− trif−/− macrophages (C) or irf3−/− macrophages (D). **, statistical difference in induction in irf3−/− macrophages compared to induction in WT macrophages (P ≤ 0.01). All experiments were performed on 3 biological replicates.

  • Fig 2
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    Fig 2

    Strain LO28 contains naturally occurring mutations in the brtA gene. (A) ClustalW alignment of the brtA gene from strains EGDe, LO28, and 10403S. Red shading indicates nucleotides not conserved between strains. The dashed line in the brtA gene in strain LO28 denotes the 188-bp deletion in this strain. Blue shading indicates the first stop codon in the brtA gene in each strain. (B) Schematic of the LMO2588 to LMO2589 region of the 10403S and LO28 genomes. Black arrows represent predicted promoters.

  • Fig 3
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    Fig 3

    IFN-β hyperinduction by strain LO28 results from constitutive mdrT derepression. (A) RT-qPCR analysis of mdrT expression in the indicated strains during mid-log-phase growth in BHI medium. *, statistical difference in mdrT expression compared to expression in strain 10403S using Student's t test (P ≤ 0.05); **, P ≤ 0.01. (B) Kinetics of mid-log-phase growth of the indicated strains in BHI medium, as measured by the OD600. Strains were back-diluted every 2 h (see Materials and Methods). (C) Kinetics of growth of the indicated strains during cytosolic infection of macrophages, as measured by a standard gentamicin protection assay. (D) RT-qPCR analysis of IFN-β induction in macrophages 4 h postinfection. *, statistical difference in IFN-β induction compared to induction by strain LO28 using Student's t test (P ≤ 0.05). All experiments were performed on 3 biological replicates.

  • Fig 4
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    Fig 4

    Overexpression of MdrT decreases strain LO28 virulence in the liver, independent of IFN-β hyperinduction. WT (A) and ifnar−/− (B) mice were infected i.v. with 1 × 104 CFU of the indicated strains. Bacterial abundance in the specified organs was determined 2 days postinfection. Black bars represent the median values. *, statistical difference in bacterial burden compared to that in mice infected with LO28 using Student's t test (P ≤ 0.05); **, P ≤ 0.01. Ten-mouse cohorts were used for each infection.

  • Fig 5
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    Fig 5

    Overexpression of MdrT in strain LO28 causes increased bile tolerance. Stationary-phase cells of the indicated genotypes were serially diluted and then spotted on BHI agar with and without supplementation with 1% porcine bile. Colony growth was visualized after 24 h at 37°C.

  • Fig 6
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    Fig 6

    Overexpression of MdrT reduces LO28 virulence in the gastrointestinal tract and gallbladder. (A) WT mice were infected i.v. with 1 × 104 CFU of the indicated strains, and bacterial abundance in the gallbladder was determined 4 days postinfection. **, statistical difference in bacterial burden compared to that in mice infected with LO28 using Student's t test (P ≤ 0.01); ND, no CFU detected. (B) WT mice were infected intragastrically with 1 × 1010 CFU of the indicated strains of L. monocytogenes, and kanamycin-resistant bacteria were enumerated in the feces at the indicated days postinfection. Black bars represent median values. No statistically significant differences in numbers of bacterial CFU were observed. Five-mouse cohorts were used for each infection.

Tables

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  • Table 1

    Strains and primers used in this study

    Strain, plasmid, or primerRelevant characteristic(s), description, or purposeSequenceaReference or source
    L. monocytogenes strains
        10403SSerotype 1/2a12
        F6854Serotype 1/2a9
        FSL J2-064Serotype 1/2b14
        LO28Serotype 1/2c3
        FSL J1-208Serotype 4a40
        AureliSerotype 4b2
        F2365Serotype 4b23
        H7858Serotype 4b10
        EGDeSerotype 1/2a13
        LO28 ΔmdrTLO28 with in-frame deletion of mdrTThis study
        LO28 + pIMK-BrtALO28 with integrated WT (10403S) allele of BrtAThis study
        LO28 + pIMKLO28 with integrated empty pIMK plasmidThis study
        LO28 ΔmdrT + pIMKLO28 ΔmdrT with integrated empty pIMK plasmidThis study
    Plasmids
        pKSV7 ΔmdrTTemperature-sensitive plasmid for allelic exchange (deletion) of mdrT11
        pIMKSite-specific integration plasmid24a
        pIMK-BrtApIMK containing WT (10403S) allele of BrtA under its own promoterThis study
    Primers
        5′ (BamHI) BrtAClones BrtA into pIMK integration vectorAAAAGGATCCACTTCCATGGTAAGGAAGGGGTCG
        3′ (NotI) BrtAClones BrtA into pIMK integration vectorAAAAGCGGCCGCTTATAAAAGCCCGTTCACACAAGCTTC
        IFN-β forRT-qPCR analysisGCACTGGGTGGAATGAGACTATTG
        IFN-β revRT-qPCR analysisTTCTGAGGCATCAACTGACAGGTC
        IL-1β forRT-qPCR analysisGACCTGTTCTTTGAAGTTGACGG
        IL-1β revRT-qPCR analysisTGTCGTTGCTTGGTTCTCCTTG
        β-actin forRT-qPCR analysisTGGCATTGTTACCAACTGGGACG
        β-actin revRT-qPCR analysisGCTTCTCTTTGATGTCACGCACG
        MdrT forRT-qPCR analysisCCCCAACCATCATTACCCGCTGAACTAAATCCGTATAG
        MdrT revRT-qPCR analysisGGTTGGATTGTGGATTCGTATGATTGGCGCG
        16S forRT-qPCR analysisACGGCTAACTACGTGCCAG
        16S revRT-qPCR analysisATGACCCTCCCCGGTTAAG
    • ↵a Underlining indicates added restriction sites.

  • Table 2

    Genes most upregulated in strain LO28 during intracellular growth compared to their expression in strain 104030Sa

    Fold increase in expressionb (log2)ORFcGene nameAnnotation
    4.7LMO0049agrDSimilar to S. aureus agrD
    4.6LMO0152Similar to oligopeptide ABC transporter-binding protein
    4.5LMO2588mdrTMajor facilitator superfamily multidrug efflux pump
    4.5LMO0204actAActin assembly protein
    4.3LMO0048agrBSimilar to S. aureus agrB
    4.1LMO2064Similar to large-conductance mechanosensitive channel protein
    4.1LMO1809plsXFatty acid/phospholipid synthesis
    4.0LMO0903osmCOsmC family protein
    3.9LMO1472dnaJHeat shock protein
    3.5LMO0135ctaXSimilar to oligopeptide ABC transporter system substrate-binding protein
    3.5LMO2433estAAcetylesterase
    3.4LMO0137Similar to oligopeptide ABC transporter, permease protein
    • ↵a The listed genes are among those determined to be statistically significantly differentially expressed by the Significance Analysis of Microarrays plug-in with a false discovery rate of 1% (see Materials and Methods).

    • ↵b In strain LO28 compared to that in strain 104030S.

    • ↵c ORF, open reading frame.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Table S1. Macrophage infection conditions to achieve similar MOIs.
      PDF file, 21K.
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Hyperinduction of Host Beta Interferon by a Listeria monocytogenes Strain Naturally Overexpressing the Multidrug Efflux Pump MdrT
Kierstyn T. Schwartz, Joshua D. Carleton, Sarah J. Quillin, Stuart D. Rollins, Daniel A. Portnoy, Jess H. Leber
Infection and Immunity Mar 2012, 80 (4) 1537-1545; DOI: 10.1128/IAI.06286-11

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Hyperinduction of Host Beta Interferon by a Listeria monocytogenes Strain Naturally Overexpressing the Multidrug Efflux Pump MdrT
Kierstyn T. Schwartz, Joshua D. Carleton, Sarah J. Quillin, Stuart D. Rollins, Daniel A. Portnoy, Jess H. Leber
Infection and Immunity Mar 2012, 80 (4) 1537-1545; DOI: 10.1128/IAI.06286-11
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