Essentiality of the UDP-galactose transporter in Cryptococcus neoformans.

Gregory A. Hill, Jr. Mentor: Tamara L. Doering, Washington University School of Medicine Department of Molecular Microbiology

Cryptococcus neoformans is a pathogenic fungus commonly found in soil and pigeon feces. In individuals with normal immune systems, the organism is easily destroyed, like many other airborne pathogens. However, in immuncompromised individuals, such as AIDS patients, the organism can cause a disease known as cryptococcosis. The disease begins as a pulmonary infection, and can advance to become a fatal meningoencephalitis.

The virulence of C. neoformans is mediated by a polysaccharide capsule that surrounds the cell. The capsule interferes with host defenses, including phagocytosis and the production of cytokines during infection. The primary focus of the Doering lab is to better understand the capsule of this organism, so that eventually a method for its destruction can be developed.

The polysaccharides that make up the capsule are synthesized from nucleotide precursors inside of the cell. My primary focus this summer has been to determine if what we believe to be a UDP-galactose transporter gene is essential to the organism. Previously, Hong Liu in the lab attempted to completely knock out the gene in these cells, but it was not possible. However, she was able to create a partial mutant. This made the lab suspect the gene was essential. I have been working with the same gene, now using diploid cells where the gene is present in two copies.

To determine the essentiality of the gene, we first use biolistic transformation to insert one copy of the fully disrupted (knock-out) gene into diploid cells. The cells then undergo sporulation on V8 media. We then select the spores by means of a secondary marker in the knock-out gene (ade2). The next step is to isolate the genomic DNA of the spores by doing genomic preps on selected colonies. After the genomic DNA is ready we use PCR with selected primers to distinguish between diploid, wild-type, and knock-out spores. We use pairs of forward and reverse primers specific to each type of cell to find out the nature of the cell. We then use agarose gel electrophoresis to visualize the results.

So far the gene seems to be essential, as at least twelve wild-type spores have been isolated, while no knock-out spores have been found. If, in fact, this gene is essential it may be an interesting drug target. In the future more research may be done to determine the actual purpose of this gene, and what processes it controls.