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Molecular Pathogenesis

ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence

Francois Lebreton, Eliette Riboulet-Bisson, Pascale Serror, Maurizio Sanguinetti, Brunella Posteraro, Riccardo Torelli, Axel Hartke, Yanick Auffray, Jean-Christophe Giard
Francois Lebreton
1Laboratoire de Microbiologie de l'Environnement, EA 956 soutenue par l'INRA, IFR 146, Université de Caen, 14032 Caen Cedex, France
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Eliette Riboulet-Bisson
1Laboratoire de Microbiologie de l'Environnement, EA 956 soutenue par l'INRA, IFR 146, Université de Caen, 14032 Caen Cedex, France
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Pascale Serror
2Unité des Bactéries Lactiques et Pathogènes Opportunistes, INRA, Domaine de Vilvert, 78350 Jouy-en-Josas, France
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Maurizio Sanguinetti
3Institute of Microbiology, Catholic University of Sacred Heart, L. go F. Vito 1, 00168 Rome, Italy
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Brunella Posteraro
3Institute of Microbiology, Catholic University of Sacred Heart, L. go F. Vito 1, 00168 Rome, Italy
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Riccardo Torelli
3Institute of Microbiology, Catholic University of Sacred Heart, L. go F. Vito 1, 00168 Rome, Italy
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Axel Hartke
1Laboratoire de Microbiologie de l'Environnement, EA 956 soutenue par l'INRA, IFR 146, Université de Caen, 14032 Caen Cedex, France
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Yanick Auffray
1Laboratoire de Microbiologie de l'Environnement, EA 956 soutenue par l'INRA, IFR 146, Université de Caen, 14032 Caen Cedex, France
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Jean-Christophe Giard
1Laboratoire de Microbiologie de l'Environnement, EA 956 soutenue par l'INRA, IFR 146, Université de Caen, 14032 Caen Cedex, France
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  • For correspondence: jean-christophe.giard@unicaen.fr
DOI: 10.1128/IAI.01218-08
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  • FIG. 1.
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    FIG. 1.

    (A) Sequence of the ace promoter region. Potential −35 and −10 region and ribosome binding site (RBS) sequences are underlined. The transcriptional start site (+1) is in boldface, and the Ers box is boxed. (B) EMSA with the promoter region of the ace gene and different concentrations (2 to 0.2 μg) of His6-ErsHTH protein. (C) EMSA of His6-ErsHTH binding to the D4-labeled DNA fragment containing the ace regulatory region. Amplifications were performed with D4-Pu and D4-Pr (Table 2). Crude cell extracts prepared from an E. faecalis Δers mutant strain (0.1 μg protein) were added to all of the reaction mixtures. Shown are the labeled ace promoter without protein (lane 1), with His6-ErsHTH (1 μg protein) (lane 2), with His6-ErsHTH and an unlabeled competitor (lane 3), and with His6-ErsHTH and a nonspecific DNA fragment (internal fragment of the ef1843 gene) (lane 4).

  • FIG. 2.
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    FIG. 2.

    Expression of ers and ace determined by Western blot or RT-qPCR analysis. (A) Western blot analysis (with antiserum against His-tagged Ers) of E. faecalis protein extracts (40 μg) from strain JH2-2 grown at 37°C (lane 2), at 46°C (lane 3), or in the presence of bile salts (lane 4) and from strain JH2-2/p3535ers (lane 5). A 0.1-μg sample of purified His6-Ers protein was loaded into lane 1. (B) RT-qPCRs experiments with the ers and ace genes and primers described in Table 2. RNAs were extracted from cells cultured under the same conditions used for protein extracts. Values that are significantly different are >2 (P < 0.05).

  • FIG. 3.
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    FIG. 3.

    Effect of ace inactivation on virulence. Percent survival of G. mellonella larvae at 16, 20, and 24 h after infection with L. lactis IL1403 (black bar), E. faecalis JH2-2 (gray bar), the JH2-2 Δace mutant (hatched bar), and the complemented Δace mutant strain (white bar). We used 6 × 106 CFU counted on an agar plate per injection. Experiments were repeated at least three times, and the results represent the mean ± standard deviation of live larvae.

  • FIG. 4.
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    FIG. 4.

    Time course of intracellular survival of E. faecalis JH2-2 (squares) and the Δace mutant (triangles) within murine peritoneal macrophages. The results shown represent the mean number ± the standard deviation of viable intracellular bacteria per 105 macrophages of three independent experiments with three wells.

  • FIG. 5.
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    FIG. 5.

    Infection with 104 cells of wild-type E. faecalis JH2-2 (•) and its isogenic Δace mutant strain (▪). Kidney pair homogenates were obtained from groups of 15 mice that were sacrificed and necropsied 48 h after a transurethral challenge. Results are expressed as log10 CFU per gram of tissue. A value of 0 was assigned to uninfected kidneys. Horizontal bars represent the geometric means.

Tables

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  • TABLE 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidRelevant characteristic(s)Reference(s) or source
    E. faecalis
        JH2-2Fusr Rifr; plasmid-free wild-type strain 9, 35
        ΔersJH2-2 isogenic Δers mutant 25
        ΔaceJH2-2 isogenic Δace mutantThis study
        Complemented Δace ace insertion in the JH2-2 Δace mutantThis study
        JH2-2/p3535ersStrain JH2-2 harboring plasmid p3535ersThis study
    E. coli
        XL1Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F′ proAB lacIqZΔM15] Tn10 (Tetr)Stratagene
        M15/pQE30ersHTHStrain harboring pQE30ersHTHThis study
    L. lactis IL 1403 4
    Plasmids
        pMADoripE194ts Emr AmprbgaB 1
        pMSP3535 3
        pGEM-T easyf1 ori lacZ AmprPromega
        pMad-ΔaceConstruction for the ace deletion mutantThis study
        pMad-aceConstruction for the complemented ace mutant strainThis study
        pMSP3535ers ers cloned into overexpression vector p3535This study
        pGEM-T-PacePromoter of ace cloned into pGEM-TThis study
  • TABLE 2.

    Primers used in this study

    Locusa or primer nameSequence (5′-3′)aUse
    ForwardReverse
    ers CGCGGATCCATAATAGATAATTGAGGTTGCTGCATCTTTTTCATGGRT-qPCR
    ef0082TTGACGTCAGCACCTTCTTCCGTAGCGTTCACCTTTGACART-qPCR
    ef0049CAGCTTTTCCGACACGTCTTTTCCCCCACGTACTTTAGCART-qPCR
    ef0782TTAGAGCGCGCAGTGAATCATGGCCGATCTGCTTCTAAART-qPCR
    ace ATGAAGGAAGCCCACAGTTGGTTGTGCCTGTTTTGGGAAGRT-qPCR
    kat GCTGTTTGGGATTTTTGGTCCATGCATATGACGGAACGACRT-qPCR
    ef1656TTTATTTGTCACCCAACCAACACAATTGAAACGGTTGATCAGAART-qPCR
    ef1664TGGCGACCTAATTACCTTGGCGTTTGCATCGTACGTGAGTRT-qPCR
    ef1816CGTCAAGACGATACCCGATTTTTCCATGTTGTGCAGGAAGRT-qPCR
    ef2185GCGATTGCTCCAACTGAATCGCCGTCCGTAATAAAAAGCART-qPCR
    ef3146CCAGAATTCTCAATTCCTCGTTCAAATTTTTGTACACCGACAGGRT-qPCR
    aceRace1ATTCCAACTAGTGCGTCTGG5′RACE PCR
    aceRace2TTGAGGTCATCTCTCCTTG5′RACE PCR
    aceRace3TCAATTCGGCGCCAAACGAA5′RACE PCR
    Prom-aceTCATTCTTTTCCTCCTTATCTCCGCAATTGGTAGAATCATTACloning in pGEM-T
    Prom-ersTAAGTTTGGTTCTGTCATTACGACTCGAAAGGAATGTTCACloning in pGEM-T
    aceDC-UbCCGGAATTCAGCTCAACTATGCCTGTCGACGCGGATCCGTCATTCCAACTAGTGCGTCCloning in pMAD
    aceDC-DbCGCGGATCCCAGCCATCCACAGAAACAACGAAGATCTTTCCTCTGCCATTAACGCGTCloning in pMAD
    p3535ersCGCGGATCCATAATAGATAATTGAGGAGTTACGCGCGCTGCAGTTAAACAATAATGTTATCTCTAATCCloning in pMSP35535
    pQE30-ersHTHGCGGATCCAAAAAGATGCAGCAAATGTTGATCGCGCGCTGCAGTTAAACAATAATGTTATCTCTAATCCloning in pQE30
    mad1F TCTAGCTAATGTTACGTTACACCloning verification
    mad2R TCATAATGGGGAAGGCCATCCloning verification
    Pu GTTGTAAAACGACGGCCAGTCloning verification
    Pr CACAGGAAACAGATATGACCCloning verification
    • ↵ a From the annotated sequence available at http://www.tigr.org .

    • ↵ b Primers used to clone upstream (U) and downstream (D) sequences of ace in order to construct a deletion mutant and complemented strain by a double-crossover (DC) event.

  • TABLE 3.

    Identification and expression of genes harboring a putative Ers box in the promoter region

    ORFdGene namePutative Ers boxExpressiona (P value) with reference to that of:
    23S rRNA genegyrA
    ef0049AACAAATGTTC-N75-ATGNDbND
    ef0782AACAAATGTTG-N272-ATG+1.01+1.1
    ef1099 ace AACAAATGTTA-N176-ATG +5.11 (0.02) +7.4 (0.016)
    ef1656AACATATGTTA-N77-ATG−1.17−1.72
    ef1664AACAATTGTTA-N132-ATG+1.12−1.01
    ef1816AACAAATGTTT-N81-ATG+1.36+1.27
    ef2185AACATATGTTT-N129-ATG—c—
    ef3146AACAATTGTTT-N424-ATG+1.99+2.4 (>0.05)
    • ↵ a Shown is the level of activation or repression in the Δers mutant compared to the wild type determined by RT-qPCR. Significant values are in bold (value > 2 and P < 0.05).

    • ↵ b ND, no detectable expression.

    • ↵ c —, Absence of the locus in the JH2-2 genome.

    • ↵ d ORF, open reading frame.

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ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence
Francois Lebreton, Eliette Riboulet-Bisson, Pascale Serror, Maurizio Sanguinetti, Brunella Posteraro, Riccardo Torelli, Axel Hartke, Yanick Auffray, Jean-Christophe Giard
Infection and Immunity Jun 2009, 77 (7) 2832-2839; DOI: 10.1128/IAI.01218-08

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ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence
Francois Lebreton, Eliette Riboulet-Bisson, Pascale Serror, Maurizio Sanguinetti, Brunella Posteraro, Riccardo Torelli, Axel Hartke, Yanick Auffray, Jean-Christophe Giard
Infection and Immunity Jun 2009, 77 (7) 2832-2839; DOI: 10.1128/IAI.01218-08
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KEYWORDS

Bacterial Proteins
Carrier Proteins
Enterococcus faecalis
Gene Expression Regulation, Bacterial
Repressor Proteins
transcription factors

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