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Fungal and Parasitic Infections

Use of Green Fluorescent Protein and Reverse Transcription-PCR To Monitor Candida albicans Agglutinin-Like Sequence Gene Expression in a Murine Model of Disseminated Candidiasis

Clayton B. Green, Xiaomin Zhao, Lois L. Hoyer
Clayton B. Green
1Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois
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Xiaomin Zhao
1Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois
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Lois L. Hoyer
1Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois
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  • For correspondence: lhoyer@uiuc.edu
DOI: 10.1128/IAI.73.3.1852-1855.2005
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    FIG. 1.

    GMS-stained (upper panels) and anti-GFP immunohistochemically stained (lower panels) tissue sections from C. albicans-infected mice. The strains used for inoculation are listed above each pair of panels. The left three panels (strains PTPI1-GFP, CAI12, and PALS1-GFP) show kidney tissue 48 h after infection. The right two panels (PALS1-GFP and PALS3-GFP) show heart tissue 24 h after infection. Inoculation of mice with the PTPI1-GFP strain produced strong, constitutive GFP production and obvious immunohistochemical staining. Staining of tissues infected with strain CAI12, which does not produce GFP, was performed as a negative control. Anti-GFP staining of strains producing GFP under the control of the ALS1 or ALS3 promoter was obvious in all kidney and heart sections in which GMS-stained fungal cells were present. Images were taken at ×400 magnification.

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  • TABLE 1.

    Observations from GMS- and immunohistochemically stained C. albicans-inoculated mouse tissue sectionsa

    Inoculation strainTime point (h)Resultb
    KidneyHeartLiver GMSSpleen GMS
    GMSIHCGMSIHC
    Uninfected48−−−−−−
    CAI1212+/+−/−+/+−/−−/+−/−
    24+/+−/−+/−−/−+/−−/−
    48+/+−/−+/+−/−+/+−/−
    PTPII-GFP12+/++/+−/−−/−−/+−/+
    24+/++/+−/−−/−+/++/−
    48+/++/+−/+−/+−/−−/+
    PALS1-GFP12+/++/+−/+−/+−/−−/−
    24+/++/++/++/++/+−/+
    48+/++/++/++/+−/−−/−
    PALS3-GFP12+/++/++/++/+−/−−/−
    24+/++/+−/+−/+−/−−/−
    48+/++/+−/−−/−−/−+/+
    PALS5-GFP12+/+−/−+/−−/−+/−+/−
    24+/+−/−+/+−/−+/−−/−
    48+/+−/−−/+−/−−/+−/−
    PALS6-GFP12−/−−/−−/−−/−−/−−/−
    24+/+−/−−/+−/−−/−−/−
    48+/+−/−−/−−/−−/−−/+
    PALS7-GFP12+/+−/−+/+−/−−/+−/−
    24+/+−/−+/+−/−+/−−/−
    48+/+−/−+/+−/−+/+−/−
    PALS9-GFP12+/+−/−−/−−/−−/−−/−
    24+/+−/−−/−−/−−/−−/−
    48+/+−/−−/−−/−+/−+/−
    • ↵ a Tissues from PALS-GFP-infected mice were analyzed using Gomori methenamine silver (GMS) staining and anti-GFP immunohistochemical (IHC) detection. Each entry in the table shows results from a pair of mice at each time point.

    • ↵ b +, presence of fungal element(s) stained black for GMS or brown for IHC visible at ×400 magnification. −, absence of fungal element(s) stained by GMS or lack of staining by IHC in the presence of GMS-positive fungal element(s). All lung sections were negative for GMS and IHC staining at all time points in all animals. All liver and spleen sections were negative by IHC staining, so only GMS results are reported in the table.

  • TABLE 2.

    Results of GFP-specific RT-PCRs from analysis of total kidney RNA from mice inoculated with C. albicans PALS-GFP reporter strains

    Duration of infection (h)Inoculation straina
    PTPI1-GFPPALS1-GFPPALS3-GFPPALS9-GFP
    12+/++/−−/−−/−
    24+/++/+−/−+/+
    48+/++/++/+−/−
    • ↵ a RNA was isolated from kidneys of animals inoculated with the specified strain, and RT-PCR was performed using GFP-specific primers. Each entry in the table shows results from a pair of mice at each time point. +, presence of RT-PCR product. −, lack of detectable RT-PCR product. Strains not listed produced all negative reactions. PALS2-GFP and PALS4-GFP reporter strains were not included in the analysis.

  • TABLE 3.

    Percent positive ALS-specific RT-PCR from analysis of total kidney RNA from mice inoculated with C. albicansa

    Duration of infection (h)% Of positive ALS-specific RT-PCR reactions
    ALS1ALS2ALS3ALS4ALS5ALS6ALS7ALS9
    12140000000
    2471869370007
    48869386000014
    • ↵ a RT-PCR was performed with ALS-specific primers (7) on total kidney RNA from the experimental mice. Each data point represents analysis of 14 mice (2 mice each inoculated with CAI12 or one of the PALS-GFP reporter strains). Percentages represent the frequency with which individual ALS-specific transcripts were detected.

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Use of Green Fluorescent Protein and Reverse Transcription-PCR To Monitor Candida albicans Agglutinin-Like Sequence Gene Expression in a Murine Model of Disseminated Candidiasis
Clayton B. Green, Xiaomin Zhao, Lois L. Hoyer
Infection and Immunity Feb 2005, 73 (3) 1852-1855; DOI: 10.1128/IAI.73.3.1852-1855.2005

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Use of Green Fluorescent Protein and Reverse Transcription-PCR To Monitor Candida albicans Agglutinin-Like Sequence Gene Expression in a Murine Model of Disseminated Candidiasis
Clayton B. Green, Xiaomin Zhao, Lois L. Hoyer
Infection and Immunity Feb 2005, 73 (3) 1852-1855; DOI: 10.1128/IAI.73.3.1852-1855.2005
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KEYWORDS

Agglutinins
Candida albicans
Candidiasis
Fungal Proteins
Fungemia
gene expression

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