By David N. Leff
Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.
A yeast fungus called Candida albicans painfully infects the cheeks of preemie babies, the vaginal tracts of millions of women and - often fatally - the heart, brain or blood of many hospitalized patients. Candida species cause more than 8 percent of all hospital-acquired infections.
The way this vicious yeast wreaks its wholesale depredations is by sheer stick-to-itiveness. Candida's fungal cells adhere tightly to the mucous membranes that line the body's epithelium, and won't let go. (See BioWorld Today, March 5, 1999, p. 1.)
If this pathogenic yeast were a bacterium or a virus, molecular microbiologists would have cloned its genes long ago. But fungi are something else. As one such scientist, William Goldman at Washington University in St. Louis, writes: "Strategies for discovering and validating virulence-related genes - that is, fungal disease implicated in disease - are largely confined to genetic approaches that were built around the molecular tools available for bacteria: mutagenesis, gene disruption, expression of cloned genes on plasmid vectors.
"The practical and genetic obstacles posed by fungal pathogens," Goldman continued, "principally the difficulties in generating defined mutants, have made the discovery of virulence genes a matter of selective guesswork." His observation appears in the current issue of Science, dated July 23, 1999, in an editorial titled, "Looking for a few good mutants." It comments on a research article in the same issue of Science, which bears the title: "An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells." Its lead author is Stanford University microbiologist Brendan Cormack.
C. glabrata is a genetic cousin of C. albicans, and responsible for its own sinister contribution to mucosal and systemic candidiasis in humans. The co-authors named their gene EPA1 (epithelial adhesin 1). It encodes a 1,034-amino-acid lectin protein, by means of which Candida grapples to its epithelial target. Finding this virulent adherence gene, the team intimates, may provide a useful target for therapies to treat such infections.
But the immediate thrust of their discovery is the strategy they deployed to defeat the yeast's genomic intractability - an end run called signature-tagged mutagenesis. Borrowing a page from the bacteriologists' hymnbook, they found their gene by creating 9,600 different mutant yeast strains, and screening them for changes in their ability to latch on to human epithelial cells.
Refractory To Genomic Analysis, Pathogenic Worms Yield To Transfection By RNA Bombing
Fungi aren't the only holdouts against molecular microbiological study. A worldwide tribe of parasitic worms called helminths also defy molecular genetic analysis. They range from roundworms, tapeworms and hookworms, which make themselves pathologically at home in the human and animal gut, to the far more pernicious helminths that cause tropical diseases such as river blindness, leischmaniasis, schistosomiasis and elephantiasis - to name a few.
Microbiologist Scott Hodgson, at Fordham University in the Bronx, N.Y., explained these worms' inaccessibility to genomic investigation. "Experimental studies on parasitic helminths," he wrote, "have been limited by a lack of parasite cell lines, and methods for molecular genetic analysis."
Would you fight these worms by bombing them? Hodgson did. He is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated July 20, 1999, titled: "Transient expression of RNA and DNA in parasitic helminths by using particle bombardment."
In this article, he pointed out at the start that "parasitic helminths cause considerable morbidity and mortality in humans [and] are an important veterinary problem. They result in significant economic losses in animal grazing and agriculture."
Hodgson and his co-authors tested particle bombardment - biolistics - as an alternative to more conventional methods of transfecting organisms. As one experimental model, they chose embryos of Ascaris, a large, heavy-bodied roundworm that can grow to a foot long or more, in the small intestine of man and beast. They collected adult female Ascaris from a local slaughterhouse, and isolated embryos from their uteri. Some 700,000 of these 5-day-old, 32-to-64-cell proto-worms were spread onto Petri dishes placed 3 centimeters below a biolistic bomber. As microcarriers for their DNA and RNA payloads, they used gold particles 1.6 micrometers in diameter. For each bombardment, these particles were coated with five-to-seven micrograms of nucleic acid plasmids.
Initially, they bombarded the embryos with an RNA gene that exists in the Ascaris genome, but earmarked to tell it apart from the endogenous sequence. Then, to gauge efficacy of transfection, they added to their construct a gene that encodes light-emitting firefly luciferase. Expression of the RNA, they reported, "occurs as early as one hour postbombardment in early embryos, and continues for at least 80 hours, the last time-point analyzed."
The team then explored the ability of biolistics to work in adult as well as embryonic helminths, and chose to test Schistosoma mansoni, the notorious pathogen of schistosomiasis. In contrast to the microscopic Ascaris, these adult worms ran 1 millimeter in diameter by a centimeter in length. Bombarded by the same regimen, "Luciferase activity derived from introduction of RNA was as high as [circa] 20-fold above background."
By Pubic Or By Scalp Hair X-Ray Diffraction, Breast-Cancer Detection Claimed Not Infallible
Physicists at France's LURE synchroton source, in Grenoble, report they can't reproduce the claim made early this year by Australian biophysicist Veronica James that X-ray diffraction of a single pubic hair can unerringly detect breast cancer. (See BioWorld Today, March 4, 1999, p. 1.)
James told BioWorld Today she preferred pubic to scalp hair, because the latter risked deformation by cosmetic permanent waving. She reported in Nature dated March 4, 1999, that "All hair samples (23 out of 23) taken from breast-cancer patients exhibited the characteristic change in their X-ray scattering patterns."
The French demurrer, published in the current Nature, dated July 15, 1999, noted, "We compared diffraction patterns of scalp hair from ten supposedly healthy people ... with the patterns from ten breast-cancer patients. ... In their experiment, "the [diffraction] ring is present in eight out of ten patterns from breast-cancer patients (instead of the 100 percent observed by James et al.)."