By David N. Leff
Singer and musician Bob Dylan (1941 - ) is known for his famous protest song, "Blowin' in the Wind." But besides its stirring lyrics, his opus packs a supremely ironical twist. Three years ago, Dylan was hospitalized with a life-threatening infection that he caught because it was literally blowing in the wind.
Mycobiologist William Goldman, professor of molecular biology at Washington University in St. Louis, tells the story: "In 1997, Dylan had to cancel a concert tour, and was hospitalized with histoplasmosis infection of the pericardium - the lining of the heart. He was treated with very powerful but very toxic antifungal drugs. Dylan told an interviewer it was the most painful thing he'd ever experienced. He made a full recovery.
"One of the problems with histoplasmosis infection," Goldman continued, "as well as with many of the other fungal infections that go deeper than the skin, is that these systemic antibiotics - ketoconazole and amphotericin B - can be quite toxic, and in fact can have effects on the patient even more serious than the burden of the pathogen. Untreated, if the organism, Histoplasma capsulatum, spreads in the body, it can be fatal - and often is."
Such is frequently the case with AIDS patients, in whom H. capsulatum is the typical defining opportunistic infection, Goldman pointed out. "It's not even a reportable disease to CDC's [Centers for Disease Control] mortality and morbidity reporting," he observed. Also, many, many people get infected with histoplasmosis each year - probably in the hundreds of thousands - whose symptoms are not serious enough to ever go to the doctor. They get a sort of a chest cold or flu-like illness. It's uncomfortable, and they're clearly sick, but then it resolves - or apparently resolves. So no one realizes that they'd gotten histoplasmosis, and then dealt with it."
Goldman's academic position at Washington University also involves something of an irony. St. Louis lies at the epicenter of Histoplasma capsulatum's pathogenic habitat. The soil mold occupies an enormous territory, embracing the valleys of the Ohio, Mississippi and Missouri rivers. In fact, it's been found underground in Maryland, Pennsylvania, New York, Texas - even Washington, D.C. - and occurs worldwide.
Some Fungi Like It Cold - And Hot
"During its life cycle," Goldman related, "H. capsulatum switches from mold to yeast. The molds are multicellular. Their main vegetative component is a long strand or filament, which contains many cells lined up in a row. They're all joined together, and will branch, like the branching of a tree. They also produce light, buoyant spores," he continued, "which can easily break away and get carried in the air as infective transport vehicles for the organism.
"The yeast phase is a unicellular form that divides by budding. It looks like a slightly smaller version of standard baker's yeast. These infectious yeasts are not good at transport, but are suited exclusively to surviving inside the host, where the temperature and moisture are higher than underground.
"Normally Histoplasma's mold-to-yeast swap is a thermal conversion," Goldman pointed out. "When the organisms are in the soil, as a mold, the ambient temperature is typically a lot lower in the environment - except in the summer in St. Louis. But when the spores are inhaled, the first thing that happens is they undergo this body-temperature shock of 37 degrees Celsius - the single most important trigger to make them convert into a yeast form."
Goldman is senior author of a paper in today's Science, dated Nov. 17, 2000. Its title: "Intracellular parasitism by Histoplasma capsulatum: Fungal virulence and calcium dependence."
"Two points," he told BioWorld Today, "end up being the most important ones to come out of our work. The technical point," he explained, "is that there had never been a way to efficiently disrupt genes in this organism. Never a way to test the role of any given gene in either its biology or pathogenesis. The technological trickery that we report in this paper is, I think, generally applicable to testing the role of any gene in Histoplasma - and very likely in related fungi, all of which have the same problem.
"The biological point," he added, "is that we've never had a defined virulence factor for this organism - partly because of the technical point. No one has had much more than inferential conclusions about what factors might be important for virulence. What this paper does is prove that one particular protein is essential for these organisms to infect mice, and to infect and kill macrophages - which is their primary host cell.
"This particular calcium-binding protein, encoded by the CBP gene, is essential for the organism's ability to survive and proliferate in very low calcium conditions, and to be a good pathogen against macrophages."
Goldman set out his strategy to counteract H. capsulatum's trickery: "The basic principle in trying to do genetic manipulation of microorganisms by molecular methods is to be able to replace a normal gene with a dysfunctional counterpart. We changed the way of presenting the CBP gene to the organism, and ended up getting a very high efficiency of targeting the recombination event that we wanted - that replaced the gene of interest with the mutated gene."
Gene KO Counter-Trickery Saved Mouse Lungs
He and his co-authors performed "a sublethal experiment" in mice they had infected with Histoplasma capsulatum. "We inoculated some of them in the nose with the wild-type virulent strain and others with the strains where we had knocked out the CBP gene. We waited about eight days, then sacrificed the animals and assayed the ability of the two types of pathogen to colonize their lungs.
"The overall results showed us that although those organisms lacking that functional gene grew just fine in culture - in mice it took more than 1,000 times as many yeast cells to start colonizing within the lungs successfully. And even then we didn't see as much lung colonization by these yeasts, as vigorous infection of the lungs, as we did when we used one-thousandth as much as the normal virulent strain.