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The sugar in a soft drink is enough to increase the number of Candida albicans cells in your mouth by a factor of 10. That's actually a small increase, one you're not likely to notice, but more vigorous growth is behind itchy fungus problems like thrush, vaginitis, and diaper rash.

Nobody ever died from diaper rash, but the mortality rates from systemic Candida infections are enormous. If unleashed in a person with a weak immune system or cancer patients on chemotherapy, the fungus – usually present on mucosal surfaces – can invade the bloodstream and cause infections anywhere in the body, killing victims 35 percent to 40 percent of the time.

The death rate from Candida is so high partly because antifungal drugs are not very effective. Since both humans and fungi are eukaryotes, many of the same drugs that kill fungi also kill human cells. One drug, amphotericin, has even been nicknamed "ampho-terrible" for its side effects. Worse, the pathogen is rapidly developing resistance to the few effective drugs.

Yet efforts to find ways to counteract resistance, or new drugs that could kill the fungi, have been stymied by the inability to understand the workings of Candida's genes.

The genetic secrets of Candida have been difficult to tease out because it does not sexually reproduce and its genome is diploid. Together, these attributes have made it impossible for geneticists to knock out random genes to find the ones critical to the cell's survival and virulence.

If the yeast were haploid, researchers would simply have to knock out the only copy of a gene. If the yeast were sexual, they could knock out one copy and then mate the cells to knock out the second copy. Since Candida is neither, researchers are stuck. The inability to randomly knock out its genes blocked simple experimental avenues that have proven invaluable in studies of other pathogens.

Instead, researchers have settled for studying the closely related but utterly benign fungus, Saccharomyces cerevisiae, in the hope that critical genes in that yeast organism will also prove critical and susceptible to drug attack in Candida. Once a gene is deemed interesting in Saccharomyces, researchers can knock out the same gene in Candida, although the process is laborious.

"But there's a very important difference between the two microbes – one can kill you and one can't," says Vincent Bruno, a student in the Graduate School of Arts and Sciences and the lab of Dr. Aaron Mitchell, professor of microbiology, whose work has now solved the problem. "Something must be different and there's a good chance those differences won't be understood by studying Saccharomyces."

Mr. Bruno, with initial help from Dr. Mitchell's former postdoc, Dr. Dana Davis, has developed a tool that can quickly and randomly knock out both copies of any gene in the Candida genome.

That tool, first rolled out in 2002, has completely revolutionized research on the pathogen. In less than a year, Mr. Bruno generated about 250 mutants, two times the number accumulated through more laborious techniques in previous decades.

"The beauty of this collection of mutants is it allows us to do things we couldn't just three to four years ago," Mr. Bruno says. "Before, people were pigeonholed into areas where there was prior knowledge from Saccharomyces. Now, we can study areas unique to Candida."

The mutants have already revealed at least one new gene related to Candida's virulence. The gene controls the pathogen's ability to transform from oval-shaped cells into hyphae, a branching medusa. The transformation, unique to Candida, is believed critical to its pathogenicity, partly because yeast cells engulfed by macrophages shoot out hyphae to break free of the immune cell.

Mr. Bruno also has used the collection to find a couple of genes important in the development of drug resistance. Only one such gene was known before.

With 7,000 genes in Candida, Mr. Bruno knows that not all the answers to its virulence are going to be solved with the 250 mutant library. Based on his initial work, however, Dr. Mitchell has received a large grant from the NIH to increase the collection.

"Vinnie and Dana really laid the groundwork for $3.5 million in new funding," Dr. Mitchell says. "At first they failed many times trying to get it to work. I know some students and postdocs think that you need to be lucky to get a good project. But Vinnie and Dana got this project and made it work through sheer perseverance."

Says Mr. Bruno, "It took some effort, but we got the ball rolling. Now it's just a matter of scaling up."

The research was supported by the Burroughs Wellcome Fund and the NIH.

—Susan Conova


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