The Role of the Ypd1 Protein in Morphogenesis in Candida albicansMegan Lindner and Dr. Daniel Herman, Research Advisor

University of Wisconsin-Eau Claire Biology Department

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 mediaYeast 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.

Results:

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.

Acknowledgements:Research sponsored by the UWEC Office of Research and Sponsorship Programs

Strain RelevantGenotype

Source/Reference

SC5314 Wildtype Fonzi and Irwin (1993)

CAI4 Δura3::imm434/Δura3::imm434

Fonzi and Irwin (1993)

YPD 11-2 ypd1/Δypd1::hisG-URA3-hisG

This work

Table 1. Candida albicans species used in this study.

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.

Spider SLAD M199 Serum

SC5314

CAI4

YPD 11-2

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.