Imagine battle armor that is so effective it continually transforms itself—adapting to protect its wearer from fast-changing assaults. In the microbial realm, the wearer is a pathogen that uses its responsive armor—consisting of the varying protein coats on its surface—to fend off the host’s immune-system defenses.
“These pathogens keep changing their coats, fooling whatever the immune system sends after them,” says HHMI international research scholar Hugo D. Luján, who studies surface-protein regulation in the intestinal parasite Giardia lamblia at his laboratory at the Catholic University of Cordoba in Argentina. Whether the pathogen defenses are compared to beasts with ever-changing scales or bandits with different disguises, the surface-protein variation poses major challenges to medicine.
Now that genomic analyses have identified the genes that express surface proteins, scientists are focusing on how pathogens detect attacks from the human immune system and quickly change their coats. That process, called antigenic variation, allows the microbe to evade the host’s immune response and extend the length of infection. And by making it difficult for the host to identify the microbial invader, it opens the door to reinfection and increases the odds that the disease will be transmitted to more human hosts.
A handful of HHMI researchers are bringing determination and creativity to the fore. A bit like high-tech tailors examining and testing surface-protein garments, they are pushing a parasite to overload its coat and reveal all its defenses, investigating how the cloak is manufactured, and studying the role of the insect vectors that transfer the parasite to the human host. Their aim is to help develop treatments or vaccines for a wide range of masters-of-disguise microbes, including those that cause African sleeping sickness, diarrheal disease, and Lyme disease (see sidebar, “Another Shot at Lyme Disease”).
The challenge is not new. A century ago, researchers experimenting with Trypanosoma brucei-infected monkeys in the laboratory of Nobel Prize winning biochemist Paul Ehrlich first uncovered evidence of antigenic variation, reporting that the trypanosomes “have acquired other biological properties … that rendered them resistant to the defensive substances.” It took another seven decades before molecular parasitologist George A.M. Cross of the Rockefeller University in New York identified the molecular basis for antigenic variation in the African trypanosome. Since then, such variation has been studied in numerous parasites as well as bacterial and viral pathogens.
Many molecular details of antigenic variation remain inscrutable, says Cross, in part because of the complex cellular mechanisms involved. Even so, he is optimistic that scientists may find ways to slow down the rate of surface-protein switching and give the human immune response more leverage to control infections.
Giardia in Argentina
At his lab in Argentina, Luján has found a way to force the Giardia parasite to reveal nearly all its surface-protein defenses at once. In doing so, he has made progress in developing a vaccine to prevent the diarrheal infection caused by the parasite and created a model for attacking other pathogens with similar antigenic talents.
Giardia—which can evade the human immune response and survive as a cyst in adverse conditions—is a common cause of parasitic gastrointestinal disease, leading to as many as 2.5 million cases of giardiasis each year in the United States. It is estimated that nearly one-fifth of the world’s population is chronically infected.
Luján became interested in Giardia’s antigenic variation while he was a postdoc in the lab of Theodore E. Nash, head of the gastrointestinal parasites section of the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland. Nash, who first reported antigenic variation in Giardia, says the Argentinian was “a real star in my lab.” Later, Luján helped reveal that, from a repertoire of about 200 genes that encode surface proteins in the Giardia genome, only one is expressed at any one time on the surface of the parasite. By the time the human immune system identifies and tries to knock out one set of surface armor, the parasite—with a wardrobe of a couple of hundred different protein armor sets—has shifted to another set.
In a December 2008 Nature paper, Luján and colleagues showed that antigenic variation in Giardia is regulated by RNA interference, a mechanism that eliminates all but one of the surface proteins at any given time. Then, in a paper in Nature Medicine in May 2010, Luján’s group reported evidence that parasites engineered to express nearly all of their surface proteins at once could be used to rally an infected host’s immune system.
Page 2 of 3
His group purified all the antigens expressed by those engineered parasites and used them to create a vaccine. They administered it orally to gerbils—first making sure the vaccine’s proteins could withstand the harsh environment of the gastrointestinal tract. It worked, successfully protecting the animals from future infections. “We were the first to demonstrate that, since the parasites continuously change their surface proteins, we must use all of the possible variants to confer full protection to subsequent infection.”
His lab has since shown that its vaccine approach works in dogs as well, and Luján is seeking a partner to try the approach on humans. In addition, he says, other researchers are now using the strategy, including a scientist at the Pasteur Institute in Paris who is investigating several candidate drugs that would promote the expression of all surface variants of the malaria parasite.
Trypanosomes in Spain
Unfortunately, the full protein exposure model is not likely to work with every pathogen. The trypanosome parasite that causes African sleeping sickness, for example, devotes a tenth of its genome—as many as 1,500 genes—to antigenic variation. NIAID’s Nash also points out that the relatively streamlined Giardia has “an extremely different mechanism” for shifting its surface proteins than the more complex trypanosome.
In the crossroads city of Granada, Spain, HHMI international research scholar Miguel Navarro is well down the path to explaining the molecular mechanisms and intricate nuclear architecture of Trypanosoma brucei, including how the parasite’s bloodstream stage expresses only one surface-protein gene at a time, the variant surface glycoprotein (VSG).
The key appears to be the dynamic association of chromosomes with structures in the parasite’s nucleus. Navarro’s laboratory at the Spanish National Research Council’s Institute of Parasitology and Biomedicine investigates “which molecules are involved in the [surface-protein] transcription switching that allows the parasite to elude the host immune response,” he says.
These pathogens keep changing their coats, fooling whatever the immune system sends after them.
Hugo D. Luján
Adopting techniques of both molecular and cell biology, Navarro uses three-dimensional microscopy and green fluorescent protein tagging to visualize the position of chromosomes in the nucleus and to investigate the position and dynamics of the telomeres—chromosome ends—that are active in antigenic variation.
The team has discovered that the African trypanosome mechanism for achieving its astounding surface variation is complex. Navarro’s recent research has focused on a protein complex—called the cohesion complex—that is essential for gluing together replicated chromosomes, or chromatids, when a cell divides. When Navarro’s group knocked out the protein complex in the trypanosome, it led to premature separation of the chromatids that contain genes for variable surface proteins. That interruption, in turn, caused a change in the antigenic switching of those proteins. These and previous findings have deepened the understanding of the trypanosome’s nuclear architecture and are helping researchers find ways to target the parasites.
Navarro first worked on antigenic variation in Cross’s laboratory from 1994 to 1998 and then moved to the University of Manchester in the U.K., where he published an influential 2001 paper in Nature. The paper reported that transcription of surface-protein genes is located in a specific area in the nucleus—called the expression site body—in such a way that only one surface gene is expressed at a time.
After returning to Spain, Navarro continued to explore the role of nuclear chromatin dynamics in antigenic variation. Cross says that Navarro’s research and related investigations are “starting to identify several genetic factors and structural attributes” of variant surface proteins that affect their switching.
“We don’t have the right tools yet to track and monitor artemisinin resistance, which is why I’ve shifted my attention on the drug-resistance side to Southeast Asia,” says Plowe. “We’re trying, in a much more accelerated fashion, what it took Tom Wellems 15 years to do in pinpointing chloroquine resistance.”
In a project that involves scientists at Oxford University, Mahido University in Thailand, and the U.S. Armed Forces Institute of Medical Sciences in Bangkok, Plowe’s lab is doing genomic studies to pinpoint gene loci that could be used as markers for that resistance. The goal is to give scientists around the world access to comprehensive data to help determine which antimalarial drugs should be used in which regions.
Page 3 of 3
“The whole discussion of malaria eradication and elimination will come to a screeching halt if artemisinin resistance spreads throughout Asia and gets to Africa,” he says. “We’re trying to develop the tools to detect that resistance and head it off.”
Tsetse Flies in the Alps
While Navarro’s work has focused on the trypanosome’s bloodstream-stage surface proteins, Isabel Roditi studies how the parasite regulates the expression of those proteins inside the gut and salivary glands of its vector, the tsetse fly. Of special interest is the parasite’s ability to sense its outside environment and respond with the appropriate adjustment of its surface coat.
“There is a complex interaction between the trypanosome and the fly as the parasites differentiate, reproduce, and migrate through different tsetse tissues,” says Roditi, an HHMI international research scholar at the University of Berne.
Using methods to genetically manipulate trypanosomes, Roditi’s team was surprised to discover that many of the large number of surface molecules on trypanosomes were not needed to grow the parasites in cultures. In addition, the researchers found that the function of the proteins became clear only when studied within the flies.
Further research led to an overhaul of the prevailing notion that abundant surface proteins, known as procyclins, were present in invariant form in every major stage of the parasite in the fly. Roditi’s lab found that procyclins are expressed only at certain times during the parasite’s progression through the fly’s gut—and not in the salivary glands.
The first vaccine that Erol Fikrig helped develop against Lyme disease targeted a surface protein of the Borrelia burgdorferei bacterium. That approach was good, but it didn’t guarantee protection against the disease. Now he’s taking a different approach: targeting a protein in tick saliva that helps the pathogen infect the host.
Gloria Rudenko, a trypanosome expert at Imperial College London, says Roditi “has given us insight into the biology of the procyclin proteins that shield the trypanosome when it is in the gut of the tsetse fly insect vector.”
Penetrating the Armor
But what do those discoveries mean to the effort to treat or prevent African sleeping sickness, a devastating disease that Roditi became aware of as a child growing up in southern Africa?
She says there is great potential to “exploit the discoveries to control trypanosome infection,” either by blocking the parasite’s reproduction in the tsetse fly or by altering the surface coat in a way that would spur an effective defense by the human host’s immune system.
Because not all surface proteins are involved directly in antigenic variation—some are transporters that acquire nutrients or enzymes that break them down, others appear to be environmental sensors—such proteins might represent potential drug targets. For example, a drug might disrupt the growth of the parasite by interfering with its ability to bind host factors; alternatively, small molecules might bind the pathogen directly and deliver spurious signals. Other approaches include hijacking nutrient transporters to introduce harmful drugs into a parasite or inhibiting the activity of certain enzymes.
Working with researcher Reto Brun of the Swiss Tropical and Public Health Institute, Roditi’s lab is now trying to develop a technique to trick trypanosomes into prematurely moving on to the next stage of their life cycle—that is, to become insect forms in their mammalian host. If parasites were to shed their VSG coat and cover themselves with a procyclin coat, the thinking goes, they would be vulnerable to destruction in the human bloodstream.
Despite the promise of finding new drugs to attack the African trypanosome, there is far less potential for developing an effective vaccine, Navarro says, because it would be so difficult to cover all 1,500 possible surface variants of the pathogen. But researchers are optimistic that they will find more effective ways to target trypanosomes and ease the burden of African sleeping sickness, which infects more than 50,000 people a year in sub-Saharan Africa and is fatal if not treated.
If scientists can make more progress in slowing the parasite’s surface-protein switching, Cross believes, “the immune response can control a trypanosome infection.” Reflecting similar optimism, Navarro hopes that “we may be able to block the mechanism that allows the parasite to escape the host immune response.”
By weakening the surface-protein armor of trypanosomes, researchers are making them vulnerable and giving hope that the age-old African threat of sleeping sickness eventually will become a bad dream of the past.