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Phenotypic Evaluation of a Candida albicans pir1/pir1 Mutant and Analysis of PIR1 Allelic Variability in a Diverse Collection of Candida albicans Isolates
RPMI 1640 Medium 90 % Germ Tube Formation YPD Medium + Serum
AsseБайду номын сангаасsment of PIR1 Allelic Variability
The presence of two different-sized alleles in strain SC5314 suggested that more allelic variants were present in the larger population of C. albicans isolates. We examined a collection of 63 oral isolates from normally healthy humans and 24 oral or anal isolates from wildlife species. Variation was restricted to the region amplified with primers PIR1C/PIR1D. Six different allelic patterns were detected. Because many of the patterns appear to be have similar-sized fragments, each was cloned and its DNA sequence determined. Four distinct alleles were present. A schematic of each allele’s repeat region is shown below in descending order by size.
pir1/pir1::PIR1-1 pir1/pir1::PIR1-2 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2
Phenotypic Testing of the pir1/pir1 Strain (continued)
pir1/pir1
pir1/pir1
PIR1A/PIR1B
PIR1C/PIR1D
PIR1G/PIR1H
Verification of Correct Strain Construction Using PCR: Genomic DNA was extracted from the wild-type control, pir1/pir1 mutant, and strains into which PIR1 alleles were reintegrated individually. As described in the previous panel, PIR1 alleles are different sizes in the repeat-containing region. Amplification with primers PIR1C and PIR1D demonstrated the two sizes. The parent strain has two alleles, the mutant has no PIR1 sequences and the reintegrant strains each have one of the two different PIR1 alleles.
60
30
C. albicans germ tubes emerging from mother yeast
0
Wild-type
Wild-type
Wild-type
650 bp 400 bp 200 bp
Germ Tube Assays: When exposed to certain environmental conditions, C. albicans forms a structure called a germ tube that is the start of hyphal growth. Germ tubes play a role in pathogenesis because cells that cannot form germ tubes show impaired virulence. Cells of the wild-type, mutant and reintegrant strains were placed into two different media that promote germ tube formation. There was no significant difference in the ability of the strains to form germ tubes (P ≤ 0.05).
Construction of a pir1/pir1 Mutant Strain
A published study suggested PIR1 is an essential gene. In other words, the authors believed that both PIR1 alleles could not be deleted without death of the cell (Martinez et al., 2004. Microbiology 150:3151-3161). Since mutant construction is difficult in C. albicans, genes are often mistakenly believed to be essential. We attempted construction of a pir1/pir1 strain and were successful. Each PIR1 allele was reintegrated individually into the mutant strain to construct control strains.
pir1/pir1
PIR1 allelic variability is attributable to the different number of copies of the repeated DNA sequences in the coding region. Strains with only one size PIR1 allele appear to result from loss of heterozygosity. For example, pattern B results from loss of the larger allele in either pattern A or C; pattern E results from loss of the smaller allele from pattern A, D or F. We also examined whether PIR1 alleles are stable in strains grown for many generations in vitro or in vivo. Passage of isolates in vitro or in vivo did not change the PIR1 allelic pattern suggesting that it is very stable.
The opportunistic fungus Candida albicans causes various forms of disease including disseminated disease, most commonly associated with immunocompromised patients, and superficial, mucosal disease that can affect immunocompromised or normally healthy individuals. C. albicans PIR1 encodes a protein associated with beta-1,3glucan in the C. albicans cell wall. Published studies suggested that Pir1 is required for proper cell wall architecture and that PIR1 is an essential gene. Our work suggested that Pir1 might contribute to adhesive interactions between C. albicans and mammalian cells. Contrary to literature predictions, we constructed a viable pir1/pir1 mutant strain. The phenotype of the strain was compared to wild-type and reintegrant control strains for growth rate, cellular morphology, germ tube formation, sensitivity to compounds that interfere with polymerization of cell wall components, and adhesion to cultured vascular endothelial cells and freshly collected buccal epithelial cells. PIR1 allelic variation was also studied. Many genes encoding C. albicans cell wall proteins have considerable allelic variability, often arising from regions of repeated sequences. Based on differences between the PIR1 alleles from strain SC5314, we developed a PCR assay and assessed allelic variability in collections of diverse C. albicans isolates from humans and wildlife. Strains passaged for many generations in vitro or in vivo were used to assess stability of the repeated sequences within PIR1 alleles. The C. albicans cell wall provides fungal cell integrity and is a structure not present in mammalian host cells. As such, the cell wall is an important antifungal drug target and its study is significant.
5 x 105 5 x 104 5 x 103 5 x 102 5 x 101 5 x 100 Wild-type pir1/pir1 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2 Wild-type pir1/pir1 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2
Xiaomin Zhao, Soon-Hwan Oh, and Lois L. Hoyer
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign
Introduction
RPMI 1640 Medium 90 % Germ Tube Formation YPD Medium + Serum
AsseБайду номын сангаасsment of PIR1 Allelic Variability
The presence of two different-sized alleles in strain SC5314 suggested that more allelic variants were present in the larger population of C. albicans isolates. We examined a collection of 63 oral isolates from normally healthy humans and 24 oral or anal isolates from wildlife species. Variation was restricted to the region amplified with primers PIR1C/PIR1D. Six different allelic patterns were detected. Because many of the patterns appear to be have similar-sized fragments, each was cloned and its DNA sequence determined. Four distinct alleles were present. A schematic of each allele’s repeat region is shown below in descending order by size.
pir1/pir1::PIR1-1 pir1/pir1::PIR1-2 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2
Phenotypic Testing of the pir1/pir1 Strain (continued)
pir1/pir1
pir1/pir1
PIR1A/PIR1B
PIR1C/PIR1D
PIR1G/PIR1H
Verification of Correct Strain Construction Using PCR: Genomic DNA was extracted from the wild-type control, pir1/pir1 mutant, and strains into which PIR1 alleles were reintegrated individually. As described in the previous panel, PIR1 alleles are different sizes in the repeat-containing region. Amplification with primers PIR1C and PIR1D demonstrated the two sizes. The parent strain has two alleles, the mutant has no PIR1 sequences and the reintegrant strains each have one of the two different PIR1 alleles.
60
30
C. albicans germ tubes emerging from mother yeast
0
Wild-type
Wild-type
Wild-type
650 bp 400 bp 200 bp
Germ Tube Assays: When exposed to certain environmental conditions, C. albicans forms a structure called a germ tube that is the start of hyphal growth. Germ tubes play a role in pathogenesis because cells that cannot form germ tubes show impaired virulence. Cells of the wild-type, mutant and reintegrant strains were placed into two different media that promote germ tube formation. There was no significant difference in the ability of the strains to form germ tubes (P ≤ 0.05).
Construction of a pir1/pir1 Mutant Strain
A published study suggested PIR1 is an essential gene. In other words, the authors believed that both PIR1 alleles could not be deleted without death of the cell (Martinez et al., 2004. Microbiology 150:3151-3161). Since mutant construction is difficult in C. albicans, genes are often mistakenly believed to be essential. We attempted construction of a pir1/pir1 strain and were successful. Each PIR1 allele was reintegrated individually into the mutant strain to construct control strains.
pir1/pir1
PIR1 allelic variability is attributable to the different number of copies of the repeated DNA sequences in the coding region. Strains with only one size PIR1 allele appear to result from loss of heterozygosity. For example, pattern B results from loss of the larger allele in either pattern A or C; pattern E results from loss of the smaller allele from pattern A, D or F. We also examined whether PIR1 alleles are stable in strains grown for many generations in vitro or in vivo. Passage of isolates in vitro or in vivo did not change the PIR1 allelic pattern suggesting that it is very stable.
The opportunistic fungus Candida albicans causes various forms of disease including disseminated disease, most commonly associated with immunocompromised patients, and superficial, mucosal disease that can affect immunocompromised or normally healthy individuals. C. albicans PIR1 encodes a protein associated with beta-1,3glucan in the C. albicans cell wall. Published studies suggested that Pir1 is required for proper cell wall architecture and that PIR1 is an essential gene. Our work suggested that Pir1 might contribute to adhesive interactions between C. albicans and mammalian cells. Contrary to literature predictions, we constructed a viable pir1/pir1 mutant strain. The phenotype of the strain was compared to wild-type and reintegrant control strains for growth rate, cellular morphology, germ tube formation, sensitivity to compounds that interfere with polymerization of cell wall components, and adhesion to cultured vascular endothelial cells and freshly collected buccal epithelial cells. PIR1 allelic variation was also studied. Many genes encoding C. albicans cell wall proteins have considerable allelic variability, often arising from regions of repeated sequences. Based on differences between the PIR1 alleles from strain SC5314, we developed a PCR assay and assessed allelic variability in collections of diverse C. albicans isolates from humans and wildlife. Strains passaged for many generations in vitro or in vivo were used to assess stability of the repeated sequences within PIR1 alleles. The C. albicans cell wall provides fungal cell integrity and is a structure not present in mammalian host cells. As such, the cell wall is an important antifungal drug target and its study is significant.
5 x 105 5 x 104 5 x 103 5 x 102 5 x 101 5 x 100 Wild-type pir1/pir1 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2 Wild-type pir1/pir1 pir1/pir1::PIR1-1 pir1/pir1::PIR1-2
Xiaomin Zhao, Soon-Hwan Oh, and Lois L. Hoyer
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign
Introduction