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Articles

Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

Sabine Keppler-Ross, Lois Douglas, James B. Konopka, Neta Dean
Sabine Keppler-Ross
1Department of Biochemistry and Cell Biology
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Lois Douglas
2Department of Microbiology, Stony Brook University, Stony Brook, New York 11794-5215
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James B. Konopka
2Department of Microbiology, Stony Brook University, Stony Brook, New York 11794-5215
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Neta Dean
1Department of Biochemistry and Cell Biology
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  • For correspondence: Neta.Dean@stonybrook.edu
DOI: 10.1128/EC.00156-10
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  • Fig. 1.
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    Fig. 1.

    High-level production of mRFP in C. albicans. (A) Yeast colony color turns from cream to pink when C. albicans (Ca) overproduces yEmRFP. CAI4 was transformed with pADH-yEmRFP and subjected to multiple rounds of selective and then nonselective growth. Note the colony color phenotype of this strain (strain SKY40) and one that contains a single integrated copy of PADH1-yEmRFP (strain SKY52) after growth on SD (−Ura) medium for 2 days at 30°C. An S. cerevisiae (Sc) strain expressing hundreds of copies of yEmRFP on 2μm plasmids is shown for comparison. (B) Fluorescence microscopy of C. albicans expressing multiple copies of yEmRFP. After multiple rounds of selective growth, almost all of the cells in the population (98 to 99%) remain brightly fluorescent after >10 generations on nonselective YPAD. (C) Elevated yEmRFP production in C. albicans containing multiple, stable copies of yEmRFP. Protein extracts were prepared from C. albicans strains with a single integrated copy of PADH1-FLAG-yEmRFP (strain SKY42) or multiple stable copies (strain SKY41), fractionated by 12% SDS-PAGE, and analyzed by Western blotting with anti-FLAG antibodies.

  • Fig. 2.
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    Fig. 2.

    Expression of yEmRFP does not affect the rate or length of hyphal formation. The kinetics and extent of hyphal formation were examined in wild-type strain BWP17 or yEmRFP-overexpressing strain SKY38. Yeast cells were grown to early logarithmic stage (1 to 2 OD600 units/ml), seeded on glass coverslips, and induced to form hyphae by the addition of 20% serum and a temperature shift to 37°C. Coverslips were removed at the indicated times and viewed by light or fluorescence microscopy. It should be noted that these panels do not represent overlapping images from the same field.

  • Fig. 3.
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    Fig. 3.

    Phagocytosis of red fluorescent C. albicans by J774 macrophages in real time. Red fluorescent C. albicans cells (strain SKY38) were added to J774 macrophages grown on coverslips in multiwell plates. Yeast cells were added at an MOI of ∼1 yeast cell per macrophage, and at various times after addition of yeast cells, CW (1 μg/ml) and PSA-FITC (20 μg/ml) were added to the culture medium to stain the surface of noningested yeast cells and macrophages, respectively. Cells were viewed by fluorescence microscopy directly, without washing, using RFP, GFP, and 4′,6-diamidino-2-phenylindole filter sets. Arrows denote RFP-positive internalized yeast cells that are inaccessible to staining with CW.

  • Fig. 4.
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    Fig. 4.

    Yeast cells are ingested more efficiently than hyphal cells by J774 macrophages. Competition assays were performed with various combinations of S. cerevisiae, C. glabrata, and C. albicans yeast or hyphal cells (preincubated for 20 min with 20% serum at 37°C to induce hyphae). Equal numbers of yeast cells expressing yEmRFP or yEGFP were mixed together and added to J774 macrophages at an MOI of 5. After 40 min, CW was added and red or green cells were scored as being in or out of macrophages. (A) Graphic representation of quantitative competition assays between nontagged C. albicans yeast cells (strain SKY51) and those expressing yEGFP (strain SKY43) that were mixed with RFP-expressing C. albicans yeast cells (strain SKY38), with S. cerevisiae yeast cells (strain SEY6210 plus pADH-yEGFP), or with C. albicans hyphal cells (strain SKY38). Sc, Ca, and Cg, S. cerevisiae, C. albicans, and C. glabrata, respectively. The percent internalization ratio was calculated by scoring yeast cells as red or green and as CW positive (outside) or CW negative (inside), as described in Materials and Methods. Representative single-focal-plane images of these assays are shown in panel B, with arrows highlighting single macrophages that have taken up both red and green yeast cells at various ratios.

  • Fig. 5.
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    Fig. 5.

    Defects in cell wall mannan but not glucan or chitin decrease the competitive uptake of yeast cells by both J774 and primary macrophages. Green fluorescent yeast strains containing mutations that affect protein mannosylation or glucan or chitin synthesis were mixed with an equal number of red fluorescent isogenic wild-type (WT) strains and coincubated with macrophages. After 40 min, CW was added and fluorescent yeast cells were visually scored as being in or out of macrophages to calculate the percent internalization ratio, as described in Materials and Methods. Data sets represent an average of three different experiments (n > 300 cells per experiment). The genotypes of the yeasts affected in N-linked mannan (och1, mnn10, hoc1), O-linked mannan (pmt1, pmt2, pmt4), β1,3-glucan (fks1), β1,6-glucan (kre1, kre6), or chitin (chs3) are listed in Table 2. Competition assays were performed using J744 cells (A) or primary bone marrow-derived macrophages (B) infected with yeast cells at an MOI of 5, as described in Materials and Methods.

Tables

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  • Table 1.

    Yeast strains used in this study

    StrainGenotypeReference or source
    C. albicans
        CAI4 ura3Δ::λimm434/ura3Δ::λimm434 19
        BWP17 ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG 50
        SKY35CAI4 and RP10::URA3This study
        SKY36BWP17 and hisG::HIS1This study
        SKY38BWP17 and PADH1-yEmRFP-URA3-ARS 25
        SKY39SKY38 and his1::HIS1This study
        SKY40CAI4 and PADH1-yEmRFP-URA3-ARSThis study
        SKY41BWP17 and PADH1-FLAG-yEmRFP-URA3-ARSThis study
        SKY42BWP17 and RP10::URA3 PADH1-FLAG-yEmRFPThis study
        SKY43BWP17 and PADH1-yEGFP-URA3-ARSThis study
        SKY51BWP17 and RP10::URA3 PADH1-yEmRFPThis study
        SKY52CAI4 and RP10::URA3 PADH1-yEmRFPThis study
        GSC1(+/−/−)CAI4 and gsc1Δ::hisG/gsc1Δ::hisG-URA3-hisG/GSC1 33
        CAP1-3121CAI4 and pmt1Δ::hisG/pmt1Δ::hisG 33
        CAP1-31CAI4 and PMT1/pmt1Δ::hisG 46
        P2-22CAI4 and PMT2/pmt2Δ::hisG 46
        CAP4-21CAI4 and PMT4/pmt4Δ::hisG 46
        CAP4-2162CAI4 and pmt4Δ::hisG/pmt4Δ::hisG 46
        NGY205CAI4 and och1ΔΔ::hisG/och1Δ::hisG 3
        SKY54CAI4 and pmt1Δ::hisG/pmt1Δ::hisG PADH1-yEmRFP-URA3-ARSThis study
        SKY55CAI4 and PMT1/pmt1Δ::hisG PADH1-yEmRFP-URA3-ARSThis study
        SKY56CAI4 and PMT2/pmt2Δ::hisG PADH1-yEmRFP-URA3-ARSThis study
        SKY57CAI4 and PMT4/pmt4Δ::hisG PADH1-yEmRFP-URA3-ARSThis study
        SKY58CAI4 and pmt4Δ::hisG/pmt4Δ::hisG PADH1-yEmRFP-URA3-ARSThis study
        SKY53CAI4 and och1Δ::hisG/och1Δ::hisG PADH1-yEmRFP-URA3-ARSThis study
    S. cerevisiae
        SEY6210 MATα ura3-52 his3-Δ200 leu2-3,112 trp1-Δ901 lys2-801 suc2-Δ9 41
        JPY12SEY6210 and mnn10Δ::LEU2 13
        VMY2SEY6210 and hoc1Δ::LEU2 35
        BY4741 MAT a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Open Biosystems
        YLR342WBY4742 and fks1Δ::KanrOpen Biosystems
        YBR023CBY4742 and chs3Δ::KanrOpen Biosystems
        YNL322CBY4742 and kre1Δ::KanrOpen Biosystems
        YPR159WBY4742 and kre6Δ::KanrOpen Biosystems
    C. glabrata
        BG88b his3Δ(1 + 631) ura3Δ(−85 + 932)::Tn903 Neor 12
        SKY60BG88b and PADH1-yEmRFP-URA3-ARSThis study
        SKY61BG88b and PADH1-yEGFP-URA3-ARSThis study
  • Table 2.

    Plasmids used in this study

    NameRelevant featuresReference or source
    CIp10 CaURA3 integrative plasmid 34
    CIp10-HIS1 CaHIS1 integrative plasmidThis study
    CIp-ADH-yEmRFP PADH1-yEmRFP in CIp10 25
    CIp-HIS1-ADH-yEmRFP PADH1-yEmRFP in CIp-HIS1This study
    CIp-ADH-FLAG-yEmRFPLike CIp-ADH1-yEmRFP but encodes N-FLAG-tagged mRFPThis study
    pADH-yEmRFP PADH1-yEmRFP in CaURA3/ARS plasmid 25
    pADH-FLAG-yEmRFPLike pADH-yEmRFP but encodes FLAG-tagged mRFPThis study
    pADH-yEGFP PADH1-yEGFP in CaURA3/ARS plasmidThis study
    YEpGAP-yEmRFP2μm URA3 plasmid containing PADH1-yEmRFP 25
    YEpGAP-yEGFP2μm URA3 plasmid containing PADH1-yEGFPThis study
  • Table 3.

    Mouse infection assay

    Yeast strainSurvival timea (days)Fungal loadb (CFU/g tissue)
    SKY40 (CAI4 Ura+ yEmRFP)2–38 × 105
    SKY35 (CAI4 Ura+)2–35 × 105
    • ↵a The survival time is based on averaged results of three separate experiments, using six mice.

    • ↵b Fungal load was determined by counting the number of viable yeast cells recovered from kidney homogenates. Kidneys (n = 6) were weighed prior to mechanical homogenization. CFU represents the number of colonies per gram of tissue and was determined by plating serial dilutions of kidney homogenates on YPAD plates and counting the number of colonies that grew after 2 days of incubation at 30°C.

Additional Files

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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - J774 macrophages incubated with C. albicans undergoing phagocytosis.
      QuickTime Movie file, 1.2MB.
    • Supplemental file 2 - J774 macrophages incubated with C. albicans undergoing phagocytosis.
      QuickTime Movie file, 2.1MB.
    • Supplemental file 3 - J774 macrophages incubated with C. glabrata undergoing phagocytosis.
      QuickTime Movie file, 3.0MB.
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Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan
Sabine Keppler-Ross, Lois Douglas, James B. Konopka, Neta Dean
Eukaryotic Cell Nov 2010, 9 (11) 1776-1787; DOI: 10.1128/EC.00156-10

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Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan
Sabine Keppler-Ross, Lois Douglas, James B. Konopka, Neta Dean
Eukaryotic Cell Nov 2010, 9 (11) 1776-1787; DOI: 10.1128/EC.00156-10
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KEYWORDS

Candida albicans
Macrophages
Mannans
Yeasts

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