Winning the War on Germs
By Joan Killough-Miller
Pamela Weathers, left, and Kristin Wobbe in the greenhouse atop Salisbury Laboratories at WPI.
Exotic new viruses and sensational flesh-eating bacteria make news headlines, but it's the more familiar microorganisms that continue to pose everyday health risks. At WPI, faculty and graduate students are leading fundamental research to understand the process of infection and maximize the availability and effectiveness of cures. Their discoveries answer questions about how plants grow and produce important compounds, and how bacteria and fungi mount an infection. They aren't trying to stave off the next epidemic or produce a new miracle drug or vaccine, but their work lays the foundation for new pharmaceuticals and prevention strategies against infectious diseases.
In one lab, the work and research of professors Pamela Weathers and Kristin Wobbe may lead to a cure for malaria, a growing world health crisis that claims at least one million lives each year, the majority of whom are children under age 5. The impact of their work could resonate globally, as more than 300 million acute cases of malaria occur each year. Reinfection is common, because the body does not develop immunity. Drug-resistant strains have already arisen in the most troubled parts of sub-Saharan Africa, Southeast Asia, and South America, rendering the common first-line antimalarial agents almost useless.
Through WPI's Interdisciplinary Plant Research Group, Weathers and Wobbe collaborate on ways to increase production of artemisinin—a plant-based compound that is currently the most effective treatment against Plasmodium falciparum, a parasite that causes malaria. Artemisinin comes from the plant Artemisia annua, sometimes called sweet wormwood or sweet annie. Chinese herbalists have used it for thousands of years, and it is considered so safe that pregnant women can take it. In addition to malaria, artemisinin is effective against certain cancers, hepatitis B, and schistosomiasis ("river blindness").
Plants are currently the only source of artemisinin, and they produce very little of the compound—a mere 1 to 2 percent of their mass. More than 700 tons of purified compound are needed yearly to treat existing cases of malaria globally. Weathers and Wobbe are exploring ways to induce sweet wormwood, either the intact plant or plant cell cultures, to produce greater quantities of artemisinin. Additionally, the WPI research team will help provide a better understanding of other terpenoid compounds, from fragrances and flavorings to the anticancer agent Taxol and many other pharmaceuticals. Their dream is to produce an artemisinin-rich plant that can be grown, picked, and eaten by anyone suffering from malaria.
In the developing world, an edible plant cure that could be cultivated locally would be a godsend. "Growing plants is something people do all over the world," says Wobbe. "All you need is water, sunshine, and some dirt. No sterile syringes, no refrigeration, and no complex extraction procedures. You get the seed, you grow the plant, you pick the thing and eat it, and you're cured." Wobbe and Weathers are working to make contact with African groups and leaders to help disseminate the cure when it is available.
Wobbe, a biochemist, works on the molecular level, trying to manipulate the genes in the pathway that synthesizes artemisinin. Weathers, a plant biologist, monitors conditions in the growth environment, trying to identify factors that optimize production. "Not too much is known about what turns these genes on and off," says Wobbe. Artemisinin, at high levels, actually appears to be toxic to the plant, which might be one reason for the low yields. However, it might be possible to shift artemisinin production to a different location within the cell, thus outwitting the plant's natural down-regulation mechanisms. Artemisinin may also be less toxic to the plant in the new cellular location.
Weathers uses Artemisia annua as a model system to study a variety of commercially useful plant products. Her culture technique involves "hairy roots"—super-producers that have been transformed with Agrobacterium, a bacteria commonly used to insert DNA in plant cells—to increase the plant hormone auxin, which stimulates multi-branched, fast-growing roots. She is also researching the optimal design of bioreactors that disperse nutrients in mist form, a more effective way to deliver precise amounts of nutrients and gases to plant tissues than immersing them in a liquid.
"What we're feeding the plants and what they sense in the environment seem to affect production in ways we can't rationalize at this point," Weathers says. "For example, different sugars give different yields. It's not just a matter of having a nutrient-rich environment or not. There are some signaling or sensing issues that are having these downstream effects." Other agents include salts, phytohormones, and "elicitors" that cause a spike in production of artemisinin. Light, oxygen, culture age, and different locations on a plant or in a reactor seem to have an effect on production as well. These factors, however, can only increase artemisinin levels tenfold at best. The combined efforts of molecular tools and bioreactor design may offer much greater yields than more traditional approaches.
"Understanding how the pathway [for artemisinin production] gets hooked together, and the communications between the different subcellular compartments has the potential to affect not just our ability to produce lots of artemisinin," Wobbe says, "but for others to take that knowledge and apply it to other compounds that are built from the same fundamental building blocks."
Reeta Prusty Rao and graduate student Brett Ericson, ECI Biotech Fellow, examine a culture dish in which colonies of yeast are growing. Prusty Rao uses the non-infectious yeast S. cerevisiae as a model system for studying the biology of pathogenic fungi.
Like that of Weathers and Wobbe, the research of colleague Reeta Prusty Rao may lead to a cure for a type of infection that has resisted existing cures.
In its benign form, yeast is an active ingredient in bread, wine, beer—the good things in life, says Prusty Rao, assistant professor of biology and biotechnology. But when "good" yeasts switch into pathogenic fungi, they can cause everything from athlete's foot to fatal lung infections. Using the noninfectious model species S. cerevisiae as a stand-in, Prusty Rao looks at how yeast transforms into its hyphal or infectious fungal form—and what can be done to stop it.
Anyone who has suffered the more common fungal infections knows how annoying—and difficult—they can be to cure. "The problem is that there is no good treatment for fungi, nothing analogous to the antibiotics we have to fight bacteria," she says. Candida albicans, the most common culprit in female vaginosis, is also the leading cause of hospital-acquired sepsis (blood-borne infection), which often invades at the site of IV lines and catheters. ("Fungi love to eat plastic," she explains.) Such infection is usually a "death knell" for postoperative or immunocompromised patients, she says.
Prusty Rao is most interested in an intercellular signaling phenomenon called quorum sensing. She explains: "One lone microbe sitting on your toe is not going to cause athlete's foot. But if there are 10,000 of them—and they know there are 10,000—they'll say, ‘How about we infect!' Quorum sensing is a mechanism for [the fungi] to count and take census before determining whether to switch to the infectious form."
Can brewer's yeast brew fuel?
An MQP student in Prusty Rao's lab would like to identify an ideal strain of yeast that could ferment ethanol automobile fuel efficiently from the sugars in an inexpensive growth medium. Through genetic manipulation, the genes needed to ferment xylose could be optimized to produce the highest possible yield. It's a long shot, says Prusty Rao, and the first step is an assay to identify the one-in-a-million yeast cell that could do the job.
Microorganisms secrete signaling molecules to announce their presence to others and measure the concentration that is taken back up into the cell to find out who else is out there. Prusty Rao is studying a molecule called indole acetic acid (IAA) to document its use as a quorum-sensing molecule in fungi. She hopes that by pinpointing the genes needed for IAA production in yeast, or by discovering other genetic factors that regulate the switch to the pathogenic form, she will identify targets for the development of new antifungal agents.
Recent work with colleague Sam Politz also holds promise for developing such treatments. They identified genes that are essential for infection to occur. When they fed mutant strains of the fungus S. cerevisiae to a nematode (small worm), they were able to show that the worms did not sicken and die, as they do when these genes are intact. This is an important breakthrough, Prusty Rao says, because this class of genes is fungus specific—that is, not shared by humans or plants. With a shared pathway, drug action that harms the pathogen also harms the host. (The existing over-the-counter remedies, which work by attacking a cholesterol-like compound in the cell membrane, are all toxic to human cells. They must be used at very low concentrations to avoid drug resistance and side effects.)
"A part of me, as a pure scientist, does not care about antifungal agents," Prusty Rao admits. "I just think that quorum sensing is a cool phenomenon and I want to decipher the mechanism of fungal pathogenesis.
"We, at WPI, are not in the pharmaceutical business," she continues. "It's their job to develop a cure. My primary goal is to do good, solid, sound research—and something good will come out of it."
Terri Camesano at Flax Pond Farm's bog, in Carver, Mass., a source of the cranberries she studies to learn more about their impact on bacterial adhesion.
Similarly, Terri Camesano's goal is genuine research. In her lab, the associate professor of chemical engineering has uncovered multiple mechanisms by which the active compounds in cranberries work on the molecular level to prevent E. coli infection—the leading cause of urinary tract infections (UTIs), among others.
UTIs affect some 8 million people each year, and it is estimated that one in five women will experience a UTI during her lifetime. E. coli is responsible for about 80 percent of cases. Antibiotic treatments exist, but bacterial resistance and side effects are a concern, making alternative strategies for treatment and prevention desirable.
Camesano's work focuses on bacterial adhesion, or "stickiness." Her findings indicate that a group of tannins called proanthocyanidins cause a number of changes that keep bacteria from attaching to—and infecting—their targets. Most fascinating is an energy barrier that surrounds the E. coli organisms and the epithelial cells of the urinary tract, making them repel like magnets with similar polarity. Measured with an atomic force microscope and converted to human scale, that repulsive force (3kT) would be enough to lift a person six feet off the ground. What's also remarkable is that the cranberry juice is acting on bacteria and the epithelial cells to keep them apart. Camesano has also demonstrated that exposure to cranberry juice makes the fimbriae—tiny tendrils that normally extend from the surface of the bacteria—collapse, making it difficult for bacteria to bind to receptors on the uroepithelium.
Don't like cranberries?
Molecules similar to the proanthocyanidins in cranberries are found in grapes, wine, tea, and other foods, but prior research has suggested that only the condensed tannins in cranberries and blueberries have the right kind of structure to produce these effects. An 8–10 ounce glass of any cranberry juice or cocktail (regular, diet, or "lite") daily is recommended for those who are prone to recurrent UTIs, and dried cranberries (sweetened or unsweetened) are thought to be beneficial as well.
Preliminary results from her experiments growing E. coli in various concentrations of cranberry juice suggest that proanthocyanidins also cause structural changes in the cell membrane of E. coli that hamper infection. The cell membrane of E. coli cells grown in cranberries showed changes in the lipopolysaccharide and peptidoglycan layers. While the impact of her findings is not yet fully understood, it appears that cranberries can "condition" the E. coli in a person's body, making it harder for the bacteria to attach to urinary tract cells. Additionally, culturing cells in cranberry juice cocktail impairs the ability of E. coli to produce IAA—the same quorum-sensing compound that Reeta Prusty Rao studies in fungal infection—which may interfere with the formation of biofilms, or communities of bacteria, thus inhibiting infection. Camesano hopes to conduct further research to assess the minimum effective dose of cranberry juice (or condensed tannins) and the necessary frequency.
Like the work of her colleagues, Camesano's research may have multiple applications. The way that E. coli attach to cells in the urinary tract is similar to some other human disease mechanisms, such as the attachment of Helicobacter pylori to the gut (in ulcers) and the attachment of Streptococcus mutans to saliva-coated surfaces in gum disease. "Cranberry compounds have been linked to antiadhesion activity that benefits each of these situations," says Camesano. "It's interesting to consider how these can have benefits in such diverse systems in the body."