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Mechanisms of Resistance

The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A

Ambre Jousselin, Caroline Manzano, Alexandra Biette, Patricia Reed, Mariana G. Pinho, Adriana E. Rosato, William L. Kelley, Adriana Renzoni
Ambre Jousselin
Infectious Diseases Service, University Hospital and Medical School of Geneva, Geneva, SwitzerlandLaboratory of Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
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Caroline Manzano
Infectious Diseases Service, University Hospital and Medical School of Geneva, Geneva, Switzerland
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Alexandra Biette
Infectious Diseases Service, University Hospital and Medical School of Geneva, Geneva, Switzerland
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Patricia Reed
Laboratory of Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
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Mariana G. Pinho
Laboratory of Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
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Adriana E. Rosato
Houston Methodist Research Institute, Houston, Texas, USA
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William L. Kelley
Infectious Diseases Service, University Hospital and Medical School of Geneva, Geneva, Switzerland
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Adriana Renzoni
Infectious Diseases Service, University Hospital and Medical School of Geneva, Geneva, Switzerland
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DOI: 10.1128/AAC.02333-15
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  • FIG 1
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    FIG 1

    Effect of prsA deletion on resistance to cefotaxime, cefaclor, cefoxitin, and ceftaroline. Spot plating population analysis profiles (spot PAP) of COL, Mu3, and their corresponding prsA deletion (ΔprsA) and prsA-complemented (ΔprsA-C) strains on MHB agar containing cefotaxime, cefaclor, cefoxitin, or ceftaroline. The upper panels correspond to MHB agar without antibiotics. The lower panels correspond to MHB agar containing antibiotic. The antibiotic concentration is indicated at the left margin. Spot serial dilutions are indicated at the right margin. The first spot, 10 μl, corresponds to 105 CFU.

  • FIG 2
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    FIG 2

    Effect of prsA on PBP profile. (A) Detection of PBP1, PBP2, PBP3, and PBP4 in membrane preparations of wild-type and ΔprsA strains. Equal amounts (20 μg) of Bocillin-FL-labeled membrane proteins were separated on a 10% SDS-PAGE gel. Fluorescently labeled PBPs are indicated by arrows. (B) Western blot analysis of PBP2 in S. aureus protein membrane extracts. PBP2 protein (80 kDa) was detected using rabbit-polyclonal anti-PBP2 antibodies. A total of 20 μg of membrane protein extract was loaded on a 10% SDS-PAGE gel. (C) Western blot analysis of PBP2A in S. aureus membrane protein extracts. PBP2A protein (70 kDa) was detected using rabbit-polyclonal anti-PBP2A antibodies. The lower panel corresponds to FtsZ antibody probing on the same PVDF membrane, used as a loading control. A total of 20 μg of membrane protein extract was loaded on a 10% SDS-PAGE gel.

  • FIG 3
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    FIG 3

    Effect of prsA deletion on oxacillin resistance in different SCCmec strain backgrounds and on mecA gene transcription. (A) Oxacillin spot plating PAPs of COL (ΔblaI ΔblaR1 ΔblaZ ΔmecR1 ΔmecI ΔmecR2), MW2 (blaI+ blaR1+ blaZ+ ΔmecR1 ΔmecI ΔmecR2), and Mu3 (ΔblaI ΔblaR1 ΔblaZ mecR1 mecI mecR2+) and their corresponding derivatives. The upper panels correspond to MHB agar without oxacillin. The lower panels correspond to MHB agar containing-oxacillin, and the oxacillin concentration is indicated at the left margin. Spot serial dilutions are indicated at the right margin. The first spot, 10 μl, corresponds to 105 CFU. (B) Steady-state levels of mecA transcript of wild-type COL strain compared to COLΔprsA and COLΔprsA-C strains, determined by qRT-PCR and normalized to 16S rRNA. Values represent the mean CT values ± the standard deviations of three independent experiments.

  • FIG 4
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    FIG 4

    Expression of PrsA and truncated PrsA derivatives. (A) Schematic representations of PrsA domain derivatives used in this study, according to Heikkinen et al. (19). Each PrsA domain derivative was introduced into to the prsA deletion strain by chromosomal insertion in the geh locus. The white box represents the first 140 N-terminal residues of PrsA containing the cysteine membrane-anchored amino acid, the gray box represents the 105 amino acid residues of the PrsA PPIase domain, and the darker gray box corresponds to the 75 amino acid residues of the C-terminal domain. The corresponding detection (+) or absence (−) of prsA mRNA or PrsA protein of each genetic construct is shown. (B) PrsA Western blot of total protein extracts from strains COL, COLΔprsA, and COLΔprsA complemented in the geh locus with wild-type prsA (COLΔprsA-C), with N- and C-terminal domains (COLΔprsA-Nter-Cter) or with PPIase domain (COLΔprsA-PPIase). Total protein extracts were run in a 15% SDS gel, and Western blotting was done with a PrsA-specific antibody (see Materials and Methods). For the detection of the PPIase domain, a longer exposure time was used.

  • FIG 5
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    FIG 5

    Effect of PrsA domains on PBP2A protein levels and oxacillin resistance. (A) The upper panel shows a PBP2A Western blot of the total protein extracts from strains COL, COLΔprsA, and COLΔprsA complemented in the geh locus with wild-type prsA (COLΔprsA-C), with N- and C-terminal domains (COLΔprsA_NterCter), or with PPIase domain (COLΔprsA_PPIase). Total protein extracts were run on a 10% SDS gel, and the membrane was probed with PBP2A- and FtsZ-specific antibodies (see Materials and Methods). The lower panel shows a quantification of three different Western blot membranes detecting PBP2A and FtsZ proteins, performed as described above. Relative abundance (AU) of PBP2A was measured by normalizing to FtsZ protein levels used as internal controls of total protein loading. An asterisk (*) denotes PBP2A protein levels significantly (P < 0.05) different from wild-type levels. (B) Oxacillin spot plating PAPs of COL, COLΔprsA, COLΔprsA-C, COLΔprsA_NterCter, and COLΔprsA_PPIase. Spot serial dilutions are indicated in the right margin. The first spot, 10 μl, corresponds to 105 CFU.

  • FIG 6
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    FIG 6

    Model of proposed PrsA posttranscriptional regulation of PBP2A following β-lactams exposure. (Step 1) β-Lactam-induced mecA and prsA transcription. The dashed arrows denote the presumed pathways leading to PrsA effects on PBP2A. PrsA protein may affect directly or indirectly PBP2A (step 2) at the translational level, and/or PBP2A protein intracellular stability (step 3), and/or (step 4) PBP2A cell membrane export/folding. The resulting PBP2A and PrsA proteins will ultimately be found active in the cell membrane compartment (49–53).

Tables

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  • TABLE 1

    Bacterial strains and plasmids used in this study

    Strain or plasmidStrainRelevant genotype or characteristicaSource or reference
    Strains
        E. coli DH5αRestriction-deficient DNA cloning strainGibco/BRL
        S. aureus
            RN42208325-4, r− m+, restriction-defective strain which accepts foreign DNA49
            COL (Tets) (cured for plasmid)AJ717HA-MRSA, bla− ΔmecR1 mecI− mecR2−50
            MW2CA-MRSA, bla+ ΔmecR1 mecI− mecR2−53
            Mu3HA-MRSA, hVISA, bla− mecR1 mecI mecR252
            CYL316Derivative of RN4220 containing the integrase for insertion of pLC84 into the lipase gene51
            Newman ΔprsAAJ3Newman prsA::kan12
            NewmanΔprsA-CbAJ4Newman prsA::kan geh::pCL84_prsA12
            MW2ΔprsAAJ724MW2 prsA::kan, Φ80α transductant of Newman ΔprsAThis study
            Mu3ΔprsAAJ457Mu3 ΔprsA, markerless deletion using pKOR1This study
            COLΔprsAAJ728COL prsA::kan, Φ80α transductant of Newman ΔprsAThis study
            COLΔprsA-CAJ760COL prsA::kan, geh::pCL84_prsA, Φ80α transductant of Newman ΔprsA-CThis study
            COL_prsA_Nterm+CtermAJ843COL prsA::kan geh::pCL84_prsA_Nterm+CtermThis study
            COL_prsA_PPIaseAJ900COL prsA::kan geh::pCL84_prsA_PPIaseThis study
            COL_prsA_Nterm+PPIaseAJ1016COL prsA::kan geh::pCL84_prsA_Nterm+PPIaseThis study
            COL_prsA_NterAJ1020COL prsA::kan geh::pCL84_prsA_NtermThis study
            COL_prsA_CtermAJ1018COL prsA::kan geh::pCL84_prsA_CtermThis study
    Plasmids
        pKOR1E. coli-S. aureus thermosensitive shuttle vector, Ampr Camr, ATc counterselection24
        pAJ342pKOR1_prsA_Mu3
        pCL84tetK; S. aureus geh locus integrating plasmid51
    • ↵a Camr, chloramphenicol resistance; Ampr, ampicillin resistance.

    • ↵b prsA-C, prsA complemented.

  • TABLE 2

    Primers used in this study

    No.NameSequence (5′–3′)aDescription
    1attB2_prsA_down-RGGGGACCACTTTGTACAAGAAGCTGGGTGCTACACAGGTGTAACAGCpKOR1-based allelic exchange
    2attB1_prsA_up-FGGGGACAAGTTTGTACAAAAAAGCAGGCATATGCCCTGCCATATCCATpKOR1-based allelic exchange
    3sacII_prsA_down-FATGCCCGCGGCACAAAACCGAGCGACCGTGGpKOR1-based allelic exchange
    4sacII_prsA_up-RATGCCCGCGGAGTTGAAACTCCTTTGTAAGpKOR1-based allelic exchange
    5upstream_primer_kpn_eco-FATGCGGTACCGAATTCTCCATATCATTTATAACAAAATAAFlanking primer for prsA site-directed mutagenesis
    6prsA_PCR_bamHI-RCGCGGATCCGAATTAAAAGATATCGGACAGATGAGFlanking primer for prsA site-directed mutagenesis
    7prsA-serine-FTTATTAGGCGCTTCTGGCGCTAGTGCCACAMutagenic primers for prsA site-directed mutagenesis (COL_prsA_serine)
    8prsA_serine-RTGTGGCACTAGCGCCAGAAGCGCCTAATAAMutagenic primers for prsA site-directed mutagenesis (COL_prsA_serine)
    9OL_pHu_pMK4_prsA_NC_down-FCTGATTCTGAAATTAAAGAACCAACAGACTTTAACAGTGAMutagenic primers for prsA site directed mutagenesis (COL_prsA_NterCter)
    10OL_pHU_pMK4_prsA_NC_up-RTCACTGTTAAAGTCTGTTGGTTCTTTAATTTCAGAATCAGMutagenic primers for prsA site directed mutagenesis (COL_prsA_NterCter)
    11New_N_signal_PPIase-FGCTTTATTATTAGGCGCTTGTGACAGCAAGAAAGCTTCACAMutagenic primers for prsA site-directed mutagenesis (COL_prsA_PPIase, COL_prsA_PPIase_Cter)
    12New_N_signal_PPIase-RTGTGAAGCTTTCTTGCTGTCACAAGCGCCTAATAAAGCMutagenic primers for prsA site-directed mutagenesis (COL_prsA_PPIase, COL_prsA_PPIase_Cter)
    13PrsA_PPIase_UAG_Bam-RGCATGGATCCCTATTTATCAGCTTTAATAATATGATMutagenic primers for prsA site-directed mutagenesis (COL_prsA_PPIase, COL_prsA_Nter_PPIase)
    14N_UAG_Bam-RGCATGGATCCTTATTTAATTTCAGAATCAGMutagenic primers for prsA site-directed mutagenesis (COL_prsA_Nter)
    15PrsA_C_N_signal-RTCACTGTTAAAGTCTGTTGGACAAGCGCCTAATAATAAAGCMutagenic primers for prsA site-directed mutagenesis (COL_prsA_Cter)
    16PrsA_C_N_signal-FGCTTTATTATTAGGCGCTTGTCCAACAGACTTTAACAGTGAMutagenic primers for prsA site-directed mutagenesis (COL_prsA_Cter)
    17PrsA-FAGTTAATGATAAGAAGATTGACGAACAAAprsA TaqMan
    18PrsA-RGAAGGGCCTTTTCAAATTTATCTTTprsA TaqMan
    19PrsA-P FAM/TAMRATGAAAAAATGCAAAAGCAATACGGCGGprsA TaqMan
    20PPIase-FTAA-AGT-TAA-ATC-TAA-GAA-AAG-CGA-CAA-AGA-AprsA TaqMan
    21PPIase-RCAA-ATT-TAC-TTG-GAT-CTT-TTG-AAA-CTT-CprsA TaqMan
    22PPIase-P FAM/TAMRAAGA-CGA-TAA-AGA-AGC-GAA-ACA-AAA-AGC-TGA-AGA-AprsA TaqMan
    23mecA_MGB_F1525TTCCACATTGTTTCGGTCTAAAATTmecA TaqMan
    24mecA_MGB_R1603AATGCAGAAAGACCAAAGCATACAmecA TaqMan
    25mecA_MGB_1553 VICCCA CGT TCT GAT TTT AAAmecA TaqMan
    • ↵a Underlined regions represent restriction enzyme sites.

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      Fig. S1 and Table S1

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The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A
Ambre Jousselin, Caroline Manzano, Alexandra Biette, Patricia Reed, Mariana G. Pinho, Adriana E. Rosato, William L. Kelley, Adriana Renzoni
Antimicrobial Agents and Chemotherapy Feb 2016, 60 (3) 1656-1666; DOI: 10.1128/AAC.02333-15

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The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A
Ambre Jousselin, Caroline Manzano, Alexandra Biette, Patricia Reed, Mariana G. Pinho, Adriana E. Rosato, William L. Kelley, Adriana Renzoni
Antimicrobial Agents and Chemotherapy Feb 2016, 60 (3) 1656-1666; DOI: 10.1128/AAC.02333-15
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