CAT Scan for Cells Permits Viewing of New Drugs' Effects on Infectious Candida

Yeast infections are familiar to most women and to some men, mostly as an easily treatable nuisance. However, in individuals who take immunosuppressive drugs or who are otherwise immunocompromised, yeast infection can sometimes spread throughout the body and become life threatening. In these instances, the infection is not easy to treat with anti-fungal drugs. The disease-causing organism also is becoming increasingly resistant to current treatments. A UCSF-led research team reports on a promising new type of anti-fungal treatment, called peptoids, in the current online edition of the journal Proceedings of the National Academy of Sciences (PNAS).

Jekyll and Hyde

One of the most common pathogens responsible for yeast infection is Candida albicans. It’s a bit of a Jekyll and Hyde. Normally it exists in a mundane-looking, one-celled, benign form. In fact, an estimated 80 percent of people harbor Candida in their genitourinary or gastrointestinal tracts. But these yeast cells can turn nasty when triggered by a changing environment or cellular milieu within the body. They transform into what the researchers describe as a “highly invasive, multi-cellular, pathogenic cell type.” The switch is accomplished by the growth of “hyphae” -- filamentous branches that reach outward from the cell body. The growing hyphae form biofilms upon which the fungus can more easily grow. Postdoctoral fellow Maho Uchida, PhD, research biophysicist Gerry McDermott, PhD, research specialist Christian Knoechel, PhD and principal investigator Carolyn Larabell, PhD, all from the UCSF Department of Anatomy, together with Mark Le Gros from Lawrence Berkeley National Laboratory, are pioneering a new type of high-resolution, three-dimensional microscopy to directly observe how potentially new drug candidates act on living pathogens, including Candida albicans. The technique is called “soft X-ray tomography.” It offers a three-dimensional window into the structure of cells. It is not used in traditional drug discovery. Typically, drug researchers target single genes or molecules within a pathogen. Then they evaluate a drug candidate’s success in blocking the function of the targeted molecule. Beyond using this gauge of initial success, they don’t learn more about targets -- or about potentially better targets -- by tracking what happens in whole cells. Soft X-ray tomography is like a CAT scan on a microscopic level. It’s used to scan individual cells in high spatial resolution. Within a cell, different organelles – little organs, if you will – absorb X-rays differently. This is the key to visualizing the insides of the cell in great detail. The X-rays are called soft because they are less energetic and penetrating than those used in a clinical or dental X-ray. The technique has become a practical tool in biomedical research within the last five years or so. Larabell is the director of the National Center for X-Ray Tomography, located at the Advanced Light Source of Lawrence Berkeley National Laboratory. The facility is the only one of its kind devoted to biochemical research. There are no microscope slides. Instead the cells are placed within a mircrocapillary tube. To keep the cells from moving, they are frozen. The tube is bombarded with X-rays. A detector collects an X-ray image, which is then stored before the tube is rotated two degrees and the procedure repeated, until the cell has been imaged from all angles. The data is run through a computer’s mathematical algorithm to reconstruct a three-dimensional image of the cells. Unlike electron microscopy, renowned for its high spatial resolution of cellular structures, soft X-ray tomography requires little sample preparation – and no dehydration, no staining, and no cutting up samples into individual slices. It only takes three minutes to obtain all the data needed to recreate three-dimensional images. Best of all, the cells are alive until the instant they are immobilized. This procedure retains the integrity of the delicate structures within the cells.

From Peptides to Peptoids

Stanford University researchers led by Annelise Barron, PhD, developed and provided two peptoids that were evaluated in the study. Peptoids are chemically similar to short protein fragments, called peptides, which humans and other organisms make naturally to fulfill various, highly specific biochemical functions within the body. Peptoids are more stable than peptides and not easily broken up by enzymes inside of us. So far, microbes have not developed drug resistance to peptides or peptoids. Using other microscopic techniques, the Stanford researchers and their previous collaborators had not been able to reach any conclusions about the mode of action of the peptoid drugs in Candida. This time, Uchida and her colleagues used soft X-ray tomography to show that peptoids prevent yeast cells from turning Jekyll-like. “Peptoids actually prevented switching to the hyphal, pathogenic form – they remain in the non-pathogenic form,” Uchida says. The experiments also reveal some bizarre cell biology in the treated yeast. A cell treated with a peptoid was more likely to form little sacs containing fat molecules, or lipids. The more disruptive of the two peptoids tested also caused the fat sacs to go where they don’t normally go. “One of the striking results was that these large lipid structures were embedded inside the nucleus,” Uchida explains. In future studies, the researchers hope to learn more about the connection between peptoid treatment, lipid production and location, and the inhibitory effect of the drugs on the growth of hyphal forms of Candida. “Nobody could really see these things in the past,” McDermott says. “We think this technique is broadly applicable in helping to accelerate new drug discovery.”

More Cells, More Techniques

Yeast cells are not the only cells that Larabell’s research group is imaging with the new technique. Thus far, the researchers have probed E. coli and Bacillus bacteria, T cells of the immune system, and red blood cells infected by the parasite that causes malaria. In addition to studying individual cells, Larabell’s lab team is working toward studying tissue samples with soft X-ray tomography. To learn more about peptoids in Candida, the researchers will be using not only soft X-ray tomography, but also a new, higher-resolution form of optical light microscopy carried out on cryogenically immobilized cells. Imaging the cells at low temperature greatly increases the lifetime of fluorescent markers used to identify the location of particular molecules inside the cell. The combination of this light microscopy technique with X-ray imaging is very powerful, Larabell says, and promises to shed new light on a number of diseases and their treatment.

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UCSF Dept. of Anatomy:Carolyn Larabell