Researchers identify new target for anti-malaria drugs

A new target for drug development in the fight against the deadly disease malaria has been discovered by researchers at MIT.

In a paper published today in the journal Cell Host & Microbe, the researchers describe how they identified the drug target while studying the way in which the parasites Toxoplasma gondii, which causes toxoplasmosis, and Plasmodium, which causes malaria, access vital nutrients from their host cells.

Around one-third of the world’s deadly infectious diseases, including malaria and tuberculosis, are caused by pathogens that spend a large portion of their life inside specially built compartments within their host cells.

These compartments, known as “parasitophorous vacuoles,” separate the host cytoplasm and the parasite by a membrane, and thereby protect the parasites from the host cell’s defenses. They also provide an environment tailored to their needs, according to Dan Gold, a postdoc who led the research in the laboratory of Jeroen Saeij, the Robert A. Swanson Career Development Associate Professor of Life Sciences in MIT’s Department of Biology.

However, the membrane of these vacuoles also acts as a barrier between the parasite and the host cell. This makes it more difficult for the parasite to release proteins involved in the transformation of the host cell beyond the membrane in order to spread the disease, and for the pathogen to gain access to vital nutrients, Gold says.

“Ultimately what defines a parasite is that they require certain key nutrients from their host,” he says. “So they have had to evolve ways to get around their own barriers, to gain access to these nutrients.”

Previous research has shown that the vacuoles are selectively permeable to small molecules, allowing certain nutrients to pass through pores in the membrane. But until now, no one has been able to determine the molecular makeup of these pores, and how they are formed.

Two new proteins

When studying Toxoplasma, the researchers discovered two proteins secreted by the parasite, known as GRA17 and GRA23, which are responsible for forming these pores in the vacuole, Gold says.

The researchers discovered the proteins’ roles by accident, while investigating how the parasites are able to release their own proteins out into the host cell beyond the vacuole membrane after invasion.

Similar research into how the related Plasmodium pathogen performs this trick had identified a so-called “protein export complex” that transports encoded proteins from the parasite into its host red blood cell, which transforms these in a way that is vital to the spread of malaria. “The clinical symptoms of malaria are dependent on this process and this remodeling of the red blood cell that occurs,” Gold says.

The researchers identified proteins secreted by Toxoplasma that appeared to be homologues, or of shared ancestry to, this protein export complex in Plasmodium. But when they stopped these proteins from functioning, they found it made no difference to the export of proteins from the parasite beyond the vacuole.

“We were left wondering what GRA17 and GRA23 actually do, if they are not involved in protein export, and so we went back to look at this longstanding phenomenon of nutrient transport,” Gold says.

When they added dyes to the host cell, and again knocked out the two proteins, the researchers found that it prevented the dyes flowing into the vacuole. “That was our first indication that these proteins actually have a role in small-molecule transfer,” he says.

More significantly though, when the researchers expressed a Plasmodium export complex gene in the modified Toxoplasma, they found that the dyes were able to flow into the vacuole once again, suggesting that this small-molecule transport function had been restored.

“All of this came together to strongly suggest that this protein that is involved in export in Plasmodium may also have an additional function in small-molecule transport,” Gold says.

Limited effects

Crucially, since these proteins are only found in the parasite phylum Apicomplexa, to which both Toxoplasma and Plasmodium belong, they could be used as a drug target against the diseases they cause, including malaria, he says.

“This very strongly suggests that you could find small-molecule drugs to target these pores, which would be very damaging to these parasites, but likely wouldn’t have any interaction with any human molecules,” he says. “So I think this is a really strong potential drug target for restricting the access of these parasites to a set of nutrients.”

In addition to malaria, the technique could also be used to target the parasite Eimeria, which affects cattle and poultry, among other animals, and therefore has a huge economic cost, Gold says.

This “very exciting” research elegantly identifies a molecular component of a pore in the vacuole that separates the growing Toxoplasma parasite from its to help in the acquisition of nutrients, according to Manoj Duraisingh, a professor of immunology and infectious diseases at Harvard School of Public Health who was not involved in the research.

“Strikingly, this molecule is conserved for this function in the related and deadly parasite Plasmodium, where it was intriguingly found to be a part of a complex required for the export of proteins for the transformation of the host red blood cell and the virulence of the parasite,” Duraisingh says. “This suggests that a virulence determinant in Plasmodium parasites was co-opted from a basic function of parasitism broadly conserved in apicomplexan .”

Researchers make progress engineering digestive system tissues

New proof-of-concept research at Wake Forest Institute for Regenerative Medicine suggests the potential for engineering replacement intestine tissue in the lab, a treatment that could be applied to infants born with a short bowel and adults having large pieces of gut removed due to cancer or inflammatory bowel disease.

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Lead researcher Khalil N Bitar, Ph.D., a professor at the institute, which is part of Wake Forest Baptist Medical Center, reported the results this week at Digestive Diseases Week in Washington, D.C. He also updated attendees on a related project to engineer anal sphincters for patients with fecal incontinence.

“Results from both projects are promising and exciting,” said Bitar. “Our latest effort, to find a new solution for the urgent need for gut-lengthening procedures, shows we can meet the basic requirements for regenerating segments of the gastrointestinal tract.”

Both projects are based on using a patient’s own cells to grow replacement tissue in the lab. Elie Zakhem, a doctoral student in Bitar’s lab, is currently working on developing tissue-engineered gut replacements. The researchers use and nerve stem cells from human intestine to engineer innervated muscle “sheets.” The sheets are then wrapped around tubular chitosan scaffolds. Chitosan is a natural biomaterial derived from shrimp shells. The material is already approved by the U.S. Food and Drug Administration for certain applications.

The tubular structures were implanted just under the skin of rats for 14 days, a first step in assessing their performance. Researchers found that the implants developed a blood vessel supply and that the tube opening was maintained. In addition, the innervated muscle “remodeled,” which means that the cells began the process of releasing their own materials to replace the scaffold.

“It is the combination of smooth muscle and neural cells in gut tissue that moves digested food material through the gastrointestinal tract and this has been a major challenge in efforts to build replacement tissue,” said Bitar. “Our preliminary results demonstrate that these cells maintained their function and the implant became vascularized, providing proof of concept that regenerating segments of the is achievable.”

The researchers’ next steps are to develop the lining of the intestine that is responsible for absorption and secretion. In a study involving research animals, they also plan to surgically connect the replacement segments to native intestine to assess function.

The group’s second project, to engineer anal sphincters, also reached a new milestone with the successful implantation of the structures in rabbits.

“These bioengineered sphincters, made with both muscle and nerve cells, restored fecal continence in the animals throughout the six-month follow-up period after implantation,” he said. “This provides proof of concept of the safety and efficacy of these constructs.”

Sphincters are ring-like muscles that maintain constriction of a body passage, such as controlling the release of urine and feces. There are actually two sphincters at the anus – one internal and one external. A large proportion of in humans is the result of a weakened internal sphincter.

“Many individuals find themselves withdrawing from their social lives and attempting to hide the problem from their families, friends and even their doctors,” said Bitar. “Many people suffer without little help.”

To engineer the internal anal sphincters, researchers used a small biopsy from the animals’ sphincter tissue and isolated that were then multiplied in the lab. In a ring-shaped mold, these cells were layered with nerve isolated from small intestine to build the sphincter. The mold was placed in an incubator, allowing for tissue formation. The entire process took about four to six weeks.

The bioengineered sphincters mimicked the architecture and function of native tissue and there are no signs of inflammation or infection after implantation. The constructs demonstrated the presence of contractile smooth muscle as well as mature nerve-cell populations.

“In essence, we have built a replacement sphincter that we hope can one day benefit human patients,” said Bitar. “Because these sphincters are made with both muscle and , they are ‘pre-wired’ to be connected with nerve pathways in the intestine.”

Bitar’s goal is to eventually conduct studies of the technology in humans. He said the technology could be applied to other diseases of the sphincter muscles, including urinary incontinence.