Transcript LindnerSpr11x

The Role of the Ypd1 Protein in Morphogenesis in Candida albicans
Megan Lindner and Dr. Daniel Herman, Research Advisor
University of Wisconsin-Eau Claire
Biology Department
Results:
Introduction:
Candida albicans is a pathogenic yeast that is able to undergo
morphogenesis, transforming from yeast to hyphal form. The dimorphism
allows C. albicans to cause systemic infection. Morphogenesis can be
triggered by limited nitrogen, neutral pH, presence of serum, and lack of a
fermentable carbon source. These environmental cues are transmitted by
several different signaling pathways to the nucleus. The Ypd-1 protein is
present in both the yeast and hyphal morphologies and could be an
intermediate used in signaling pathways that promote morphogenesis.
Disrupting the gene and then phenotypic characterization will show
whether the Ypd-1 protein is actually an intermediate in these signal
transduction pathways. The ura-blaster technique was used to disrupt the
YPD-1 gene. Then gene disruption was confirmed by Southern blot
analysis. We have created a C. albicans YPD-1 heterozygote strain and are
currently working on creating a null mutant. The strains will then be
examined for defects in morphogenesis using phenotypic characterization
media.
Methods:
The ura-blaster technique was used to disrupt the YPD-1 gene creating a
heterozygote. Gene disruption was confirmed by Southern blot analysis.
The heterozygote phenotype was characterized using the following media
that would promote hyphae formation:
-Spider media
-SLAD media
-M199 media at pH 7.5
-Serum media
Yeast cultures of the heterozygote were grown overnight in a 5mL culture
of YNB broth. Media was inoculated with a volume that would produce
50-100 colonies per plate and the plates were incubated at 37 degrees
Celsius for seven days before the pictures were taken.
Strain
Relevant
Genotype
Wildtype
Source/
Reference
SC5314
Fonzi and Irwin
(1993)
CAI4
Δura3::imm434/ Fonzi and Irwin
Δura3::imm434 (1993)
YPD 11-2 ypd1/Δypd1::his This work
G-URA3-hisG
Table 1. Candida albicans species used in this study.
Spider
SLAD
M199
Serum
SC5314
CAI4
YPD 11-2
Figure 1. Plate cultures of SC5314 (wildtype), CAI4, and YPD 11-2 heterozygote.
Strains were inoculated on spider, SLAD, M199, and serum media and incubated for
seven days. All strains underwent morphogenesis except YPD 11-2 on spider media.
Discussion:
YPD 11-2 was able to undergo morphogenesis when grown on solid media with
limited nitrogen (SLAD), increased pH (M199), and in the presence of serum
(serum). However, the YPD 11-2 heterozygote was unable to undergo
morphogenesis on media lacking a fermentable carbon source (spider). Work is
underway to create a null mutant strain in which both YPD1 alleles have been
disrupted. The null mutant strain will be analyzed for additional defects in
morphogenesis. In addition, yeast two-hybrid analysis will be conducted to
identify proteins that interact with the Ypd1 protein.
References:
Calear J, Herman D, Calderone R. Identificantion of YPD1, a gene of Candida
albicans which encodes a two-component phospho-histidine intermediate.
Yeast 2000; 16(11): 1053-1059.
Pfaller MA, Diekema DJ. Epidemiology of invasive Candidiasis: a persistent
public health problem. Clinical Microbiology Reviews 2007; 20(1): 133-163.
Acknowledgements:
Research sponsored by the UWEC Office
of Research and Sponsorship Programs