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MOLECULAR AND CELLULAR PATHOGENESIS

The Legionella pneumophila icmGCDJBFGenes Are Required for Killing of Human Macrophages

Mary Purcell, Howard A. Shuman
Mary Purcell
Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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Howard A. Shuman
Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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DOI: 10.1128/IAI.66.5.2245-2255.1998
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  • Fig. 1.
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    Fig. 1.

    (A) Intracellular multiplication of strain LELA3896 within HL-60-derived macrophages. Differentiated HL-60 cells (1.5 × 106 per well) were infected with approximately 104 bacteria on day 0. The number of CFU of each strain per milliliter was determined daily for 5 days, in triplicate, on ABCYE plates. ▪, JR32; □, LELA3896; •, LELA3896(pMMB207); ○, LELA3896(pMW100). Error bars indicate the standard error of the mean. (B) Cytotoxicity of strain LELA3896 for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the absorbance at 570 nm (A570). ▪, JR32(pMMB207); □, JR32(pMW100); •, LELA3896; ○, LELA3896(pMMB207); ▴, LELA3896(pMW100). Values are the average of three determinations. Error bars indicate the standard error of the mean.

  • Fig. 2.
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    Fig. 2.

    (A) EcoRI restriction map of the genomic insert on plasmid pMW100. EcoRI restriction sites are labeled R. The approximate size of each EcoRI restriction fragment is shown in kilobases. Ptac of the pMMB207 vector directs transcription to the left into the 5.5-kbEcoRI fragment. The dotted line at the 3′-end of the 5.5-kbEcoRI fragment represents the region not sequenced. Mak− DNA hybridization groups represented on the genomic insert of pMW100 as shown by Southern analysis are shown above theEcoRI fragments. (B) ORFs present on the genomic insert of pMW100 and locations of Tn903dIIlacZ insertions (▹) and deletion substitutions (▪) in the Mak−mutants. Triangles on top of one another indicate that the LELA strains contain a Tn903dIIlacZ insertion at the same nucleotide. Vertical numbers indicate the strain number. ORFs shown were identified by nucleotide sequencing and MacDNAsis analysis. Solid arrows indicate the direction of transcription of each ORF. The location with respect to each EcoRI restriction site and size of each ORF is approximate. icmE is represented as a partial ORF, as indicated by a dotted 5′ end.

  • Fig. 3.
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    Fig. 3.

    Cytotoxicity of the icmF mutant strains for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 cells per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A570. (A) icmF mutant strain LELA1275. ▪, JR32(pMMB207); □, LELA1275(pMMB207); •, LELA1275(pMW100). (B) icmF mutant strain LELA1718. ▪, JR32(pMMB207); □, LELA1718(pMMB207); •, LELA1718(pMW100). Values are the average of three determinations. Error bars indicate the standard error of the mean.

  • Fig. 4.
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    Fig. 4.

    Representation of predicted transmembrane domains in theicm gene products. The hydropathic profiles of the predicted Icm proteins are shown according to the Kyte-Doolittle scale with a window size of 20 amino acids. The transmembrane domains as identified by the Psort program are represented as bars above the graphs. The vertical axis represents the hydrophobic value as measured on the Kyte-Doolittle scale, and the horizontal axis represents the number of amino acids in the icm gene products.

  • Fig. 5.
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    Fig. 5.

    Cytotoxicity of the icmB mutant strains containing different plasmids for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A570. (A) icmBmutant strain LELA3393. ▪, JR32(pMMB207); □, LELA3393(pBC SK+); •, LELA3393(pMW100); ○, LELA3393(pMW560). (B) icmBmutant strain LELA1012. ▪, JR32(pMMB207); □, LELA1012(pBC SK+); •, LELA1012(pMW100); ○, LELA1012(pMW560). (C) icmBmutant strain LELA1223. ▪, JR32(pMMB207); □, LELA1223(pBC SK+); •, LELA1223(pMW100); ○, LELA1223(pMW560). (D) icmBmutant strain LELA3896. ▪, JR32(pMMB207); □, LELA3896(pBC SK+); •, LELA3896(pMW100); ○, LELA3896(pMW560). Values are the average of three determinations. Error bars indicate the standard error of the mean.

  • Fig. 6.
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    Fig. 6.

    Cytotoxicity assay of L. pneumophila strains containing different plasmids for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 cells per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A570. (A) icmJallelic exchange strain MW656. ▪, JR32(pMMB207); □, MW656(pMMB207αb); •, MW656(pMW100); ○, MW656(pMW680);Embedded Image, MW656(pMW681); ▵, MW656(pMW560); ▾▴, MW656(pMW560, pMW790). (B) icmD mutant strain LELA1205. ▪, JR32(pMMB207); □, LELA1205(pMMB207αb); •, LELA1205(pMW100); ○, LELA1205(pMW734); Embedded Image, LELA1205(pMW736). (C) icmC allelic exchange strain MW645. ▪, JR32(pMMB207); □, MW645(pMMB207αb); •, MW645(pMW100); ○, MW645(pMW604); Embedded Image, MW645(pMW728); ▵, MW645(pMW730); ▾▴, MW645(pMW734). (D) icmG allelic exchange strain MW635. ▪, JR32(pMMB207); □, MW635(pMMB207αb); •, MW635(pMW100); ○, MW635(pMW741); Embedded Image, MW635(pMW743). Values are the average of three determinations. Error bars indicate the standard error of the mean.

Tables

  • Figures
  • Table 1.

    Bacterial strains used in this study

    StrainGenotype and featuresReference or source
    L. pneumophila
     JR32NaCl-sensitive isolate of AM51128
     LELA1205JR32icmD1205::Tn903dIIlacZ, DNA hybridization group II28
     LELA1506JR32icmB1506::Tn903dIIlacZ, DNA hybridization group II28
     LELA1984JR32icmE1984::Tn903dIIlacZ, DNA hybridization group II28
     LELA1996JR32icmB1996::Tn903dIIlacZ, DNA hybridization group II28
     LELA2517JR32icmC2517::Tn903dIIlacZ, DNA hybridization group II28
     LELA3244JR32icmD3244::Tn903dIIlacZ, DNA hybridization group II28
     LELA3379JR32icmB3379::Tn903dIIlacZ, DNA hybridization group II28
     LELA3393JR32icmB3393::Tn903dIIlacZ, DNA hybridization group II28
     LELA3563JR32icmB3563::Tn903dIIlacZ, DNA hybridization group II28
     LELA3896JR32icmB3896::Tn903dIIlacZ, DNA hybridization group II28
     LELA1223JR32icmB1223::Tn903dIIlacZ, DNA hybridization group IV28
     LELA1566JR32icmB1566::Tn903dIIlacZ, DNA hybridization group IV28
     LELA3150JR32icmB3150::Tn903dIIlacZ, DNA hybridization group IV28
     LELA3323JR32icmB3323::Tn903dIIlacZ, DNA hybridization group IV28
     LELA1012JR32icmB1012::Tn903dIIlacZ, DNA hybridization group VI28
     LELA2947JR32icmB2947::Tn903dIIlacZ, DNA hybridization group VI28
     LELA1275JR32icmF1275::Tn903dIIlacZ, DNA hybridization group XVII28
     LELA1718JR32icmF1718::Tn903dIIlacZ, DNA hybridization group XVII28
     MW635JR32icmG::KmrThis study
     MW645JR32 icmC::KmrThis study
     MW656JR32icmJ::KmrThis study
     MW627JR32 tphA::KmrThis study
     Philadelphia-1Virulent, serogroup 116
    E. coli
     DH5αF−endA1 hsdRI7(r−k m+k) supE44 thi-1 λ− recA1 relA1 Δ(argF-lacZYA) U169 φ80dlacZΔM15 deoR gyrA96Nalr33
     LE392F− e14− (McrA−)hsdR514 (r−km+k) supE44 supF58 lacY1 or Δ(lacIZY)6 galK2 galT22 metB1 trpR553
     LW211LE392 with an integrated RP4 (Δbla tetA::Mu) transferred by P1 transduction from SM10; Tra+ KmrApr Tcs28
     MW67DH5α containing a L. pneumophilaPhiladelphia-1 chromosomal DNA library consisting of partialEcoRI fragments (10–20 kb) in pMMB207This study
  • Table 2.

    Plasmids used in this study

    PlasmidGenotype and featuresReference or source
    pAB13Tn903dIIlacZ-containingEcoRI fragment from LELA1012 in pBSK KS+28
    pAB14Tn903dIIlacZ-containingEcoRI fragment from LELA1566 in pBSK KS+28
    pBluescript II KS (+)oriR (f1) MCS oriR (ColE1), AprStratagene
    pBC SK (+)oriR (f1) MCS oriR (ColE1), CmrStratagene
    pBR322oriR (ColE1), AprTcrNew England Biolabs
    pCR2.1oriR(f1) MCS oriR (ColE1), Apr KmrInvitrogen
    pMMB207oriR (RSF1010), Cmr25
    pMMB207αbpMMB207 MCS, Cmr30
    pMMB207αb::GmpMMB207 MCS, GmrThis study
    pMW100icmEGCDJB-, tphA-, andicmF-containing fragment in pMMB207This study
    pMW150Tn903dIIlacZ-containingEcoRI fragment from LELA3563 in pMMB207This study
    pMW152Tn903dIIlacZ-containingEcoRI fragment from LELA1223 in pMMB207This study
    pMW318Tn903dIIlacZ-containingEcoRI fragment from LELA1996 in pMMB207This study
    pMW320Tn903dIIlacZ-containingEcoRI fragment from LELA1506 in pMMB207This study
    pMW4201,864-bp icmJ-containing fragment in pBSK KS+This study
    pMW4242,892-bp icmGCD-containing fragment in pBSK KS+This study
    pMW5253,257-bpHindIII fragment from pMW100 in pBSK KS+This study
    pMW528icmB-containing fragment in pBSK KS+This study
    pMW560icmB-containing fragment in pBC SK+This study
    pMW564897-bp icmC-containing fragment in pCR2.1This study
    pMW565811-bpicmD-containing fragment in pCR2.1This study
    pMW5661,495-bp icmG-containing fragment in pCR2.1This study
    pMW5762,004-bp tphA-containing fragment in pUC18This study
    pMW582pMW424 with aKpnI site at bp 2993This study
    pMW5842,540-bpicmGCD-containing fragment in pUC18This study
    pMW587pMW420 containing ΔicmJ with aSalI site at bp 2993This study
    pMW589pMW576 containing ΔtphA with a SalI site at bp 6862This study
    pMW591pMW584 containing ΔicmGwith a SalI site at bp 995This study
    pMW593pMW584 containing ΔicmC with aSalI site at bp 1831This study
    pMW596pMW589 with Km cassette inserted in ΔtphAThis study
    pMW598pMW591 with Km cassette inserted in ΔicmGThis study
    pMW600pMW593 with Km cassette inserted in ΔicmCThis study
    pMW6021,331-bpHindIII-BamHI fragment from pMW587 in pUC18This study
    pMW6042,540-bp icmGCD-containing fragment in pMMB207This study
    pMW606pMW602 with Kmr cassette inserted in ΔicmJThis study
    pMW616ΔtphA::Kmr in pLAW344This study
    pMW618ΔicmG::Kmr in pLAW344This study
    pMW620ΔicmC::Kmr in pLAW344This study
    pMW622ΔicmJ::Kmr in pLAW344This study
    pMW6801,864-bp icmJ-containing fragment in pBC, opposite orientation to PlacUV5This study
    pMW6811,864-bpicmJ-containing fragment in pBC, same orientation to PlacUV5This study
    pMW728897-bpicmC-containing fragment in pMMB207αb, same orientation to PtacThis study
    pMW730897-bpicmC-containing fragment in pMMB207αb, opposite orientation to PtacThis study
    pMW734811-bpicmD-containing fragment in pMMB207αb, same orientation to PtacThis study
    pMW736811-bpicmD-containing fragment in pMMB207αb, opposite orientation to PtacThis study
    pMW7411,495-bpicmG-containing fragment in pMMB207αb, same orientation to PtacThis study
    pMW7431,495-bpicmG-containing fragment in pMMB207αb, opposite orientation to PtacThis study
    pMW7901,894-icmJ-containing fragment in pMMB207αb::Gm, opposite orientation to PtacThis study
    pLB41HindIII fragment containing Gmr in pBR322L. Babiss and D. Figurski
    pUC18oriR(ColE1) MCS, AprNew England Biolabs
  • Table 3.

    Characteristics of predicted ORFs

    Potential ORF identifiedNo. of amino acidsaMol mass (kDa)bPredicated cellular locationcPredicted motifsdPredicted homologye
    icmE
    icmG26929.7IM (1 TM)NoneNone
    icmC19420.3IM (4 TM)NoneNone
    icmD13213.5IM (1 TM)NoneNone
    icmJ21224.2IM (1 TM)NoneNone
    icmB1,009112.1IM (1 TM)ATP/GTP binding siteNone
    tphA41846.4IM (12 TM)Sugar transport proteinProP
    icmF973110.7IM (2 TM)ATP/GTP binding siteNone
    • ↵a The number of amino acids encoded by each complete ORF is reported. icmE is partial on pMW100; therefore the total number of amino acids is not reported.

    • ↵b The predicted molecular mass of each complete ORF is shown. The molecular mass of the partial icmE cannot be determined.

    • ↵c The location of each ORF was predicted by the Psort program (26). IM designates an inner membrane location. The number of transmembrane domains (TM) as determined by the Psort program is shown in parentheses.

    • ↵d Motif searches were performed with the GCG motif search program.

    • ↵e Homology searches were performed at the amino acid and nucleotide levels by using the TFASTA, BLASTP, and BLASTX (18) programs.

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The Legionella pneumophila icmGCDJBFGenes Are Required for Killing of Human Macrophages
Mary Purcell, Howard A. Shuman
Infection and Immunity May 1998, 66 (5) 2245-2255; DOI: 10.1128/IAI.66.5.2245-2255.1998

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The Legionella pneumophila icmGCDJBFGenes Are Required for Killing of Human Macrophages
Mary Purcell, Howard A. Shuman
Infection and Immunity May 1998, 66 (5) 2245-2255; DOI: 10.1128/IAI.66.5.2245-2255.1998
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KEYWORDS

Genes, Bacterial
Legionella pneumophila
macrophages

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