October 30, 1997
Harvard
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  Genetic Secrets of Killer Fungus Found

By William J. Cromie

Gazette Staff

Imagine a Halloween creature that grows inside the cells of human organs. Normally, it takes the form of a relatively harmless microball slipping easily through blood vessels. But at its destination, it transforms itself into a snakelike form that bores holes through cells, killing them.

Julia Koehler, a Harvard fellow in infectious diseases, actually made a movie of this treat-or-trick, Jekyll-and-Hyde organism that lives inside some 40 million people -- more than 15 percent of the people in the United States. When a person's immune system works as it should, gentle Jekyll causes no harm. But when things go wrong, horrible Hyde causes infections that range from irritating to lethal.

Koehler and Hsiu-Jung Lo, her colleague at the Whitehead Institute for Biomedical Research in Cambridge, uncovered the genetic signaling responsible for this transformation and how it might be blocked with new types of drugs.

Known scientifically as Candida albicans, "it is, by far, the most predominant fungus involved in human disease," Koehler says. "Fungal infections acquired in U.S. hospitals doubled from 1980 to 1990, and Candida was responsible for almost 60 percent of them. It killed one of every three people with a bloodstream infection."

Usually, Candida lives harmlessly in the mucous membranes of the mouth, vagina, penis, and gut. Its infectious predilections are harnessed by helpful bacteria and protective white blood cells. When overuse of antibiotics kills the bacteria, or medical treatments like chemotherapy interfere with the operation of the immune system, Candida multiplies and spreads.

It can cause localized infections of the vagina or penis, and thrush in the mouths of young children. But it becomes life-threatening in people whose immune system is severely compromised: premature infants, those who undergo intensive surgery or are severely burned, and cancer and AIDS patients treated with chemotherapy or bone marrow transplants.

A Boring Shape

Koehler, a pediatrician, became interested in understanding Candida as a result of her work with premature babies and young children in intensive care units.

Researchers knew the fungus changed shape to become virulent, but they had no idea how Candida did it. The Jekyll form consists of single round or elliptical cells, a shape that the fungus assumes when it lives in its noninfectious stage in mucous membranes.

Humans have both protective white blood cells called neutrophils and macrophages that engulf and chew up single Candida cells and other invading microbes. In the case of a weakened immune system, however, these microbe-eaters get overwhelmed.

Inside macrophages and internal organs, the single cells join together to form long filaments called hyphae. This is the shape Candida assumes when it invades the organs of severely ill people -- the shape of Hyde.

Koehler's movie clearly shows hyphae boring through the walls of macrophages. They resemble threadlike snakes that wiggle out of these weakened cells, slip through blood vessels or lymph ducts, then force their way into the cells of other tissues and organs.

Antifungal drugs against Candida have only limited effectiveness. The most effective one comes with such toxic side-effects that many patients quit taking it. A less sickening drug exists, but Candida often develops resistance to it.

"Numerous drugs exist for bacteria, but we have only two for invasive Candida," Koehler says. "We need to understand how the fungus switches forms so we can develop new ways to fight it."

One approach involves gene knockouts. Researchers knock out, or mutate, genes they believe to be responsible for a microbe's virulence. If offspring of the bug lack the ability to produce disease, this means that the mutated gene lies at the core of making the microbe infectious. If one gene doesn't do it, they knock out others.

In this way, scientists can both learn how an organism works and can locate targets for drugs.

Such experiments are particularly difficult with Candida, however. Fortunately, Gerald Fink of the Whitehead Institute spent many years studying the genetics of a related fungus, Saccharomyces cerevisiae, the yeast that makes bread rise. He discovered that this yeast also contains a genetic switch that can transform its cells from individual grain-like bodies to a filamentous form.

The genes of these two fungi are so alike that finding how to turn off the shape switch in one should work in the other. Once the switch is off, the researchers reasoned, Jekyll could not turn into Hyde.

Double Whammy Needed

When the team found a suspect gene in baker's yeast, Koehler made a strain of Candida with the equivalent gene mutated so it wouldn't function. The result was disappointing, however. The fungal cells made many fewer hyphae, but these dangerous prowlers weren't completely eliminated.

After thinking about it, the researchers reasoned there must be another genetic circuit involved in the Jekyll-Hyde transformation. Koehler's colleague, Hsiu-Jong Lo, knocked out a second gene in Koehler's mutated Candida. That double whammy did the trick.

Candida cells injected into mice usually kill them. But mice injected with the doubly mutated fungal cells survive.

"When you inject Candida into animals' bloodstreams and they do not die, you know you're on the right track," Koehler says. "But you still fall short of knowing exactly what happens in the animals' bodies."

To get these details, she put macrophage cells from mice under a microscope and "fed" them "wild" and mutated Candida. A movie made from microscope photos, taken every 15 to 20 seconds, clearly shows single wild cells building themselves into snakelike filaments and tearing out of the white blood cells.

Mutated strains of Candida, in contrast, stay put. You see chewed-up fungi fragments retained within the walls of the white cells. Presumably, the same thing would happen in human cells.

Seeking the Cure

The next step is to go from stopping Candida infections in mice cells under a microscope to developing drugs that can do the same thing in humans infected with the fungus.

Lo is optimistic about when this can be done. "It's in the process now," she says, waving her hand across the laboratory. "Things look positive."

Lo also believes this path can be followed to find cures for other fungal infections in both humans and in food crops, where these microbes take a high toll. Many fungi go through the same Jekyll and Hyde transformation, she points out. Researchers at Purdue University are already working on knocking out analogous genes in a fungus that attacks rice plants.

Koehler is more cautious. "Many questions have to be answered before we can develop new antifungal drugs for humans," she insists. "For example, we need to know more about how Candida moves from the gut, its main point of entry, into the blood, and out again into other organs and tissues."

She also is interested in the switch from Hyde back to Jekyll. Mutant Candida stuck in the filamentous form produce less disease in the vaginas and mouths of mice. Perhaps, Koehler notes, "it's the ability to switch back and forth between single, round cells and long filaments, rather than filamentous growth per se that makes Candida albicans such a killer."

 


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