New research led by University of Pennsylvania scientists has revealed how the parasite Toxoplasma gondii crosses the blood-brain barrier.
Long before Zika became a grave concern of expecting mothers, a parasitic infection was terrifying not only pregnant women but individuals with compromised immune systems, such as patients on cancer therapies or with HIV/AIDS. Moreover, as alluded to in the previous statement, Toxoplasma gondii can be passed along from mothers to the fetus, putting the babies at risk of severe neurological disease.
Scientists have known for many years that T. gondii can affect the brain, even influencing the behavior of its hosts. However, some have debated the exact mechanisms of how the parasite crosses the blood-brain barrier, a physical obstacle intended to keep pathogens out of the brain.
Researchers at the University of Pennsylvania School of Veterinary Medicine, along with colleagues from across the country, have identified how the parasite makes its way into the brain. Using a powerful imaging technique that allowed the scientists to track the presence and movement of parasites in living tissues, the researchers found that Toxoplasma infects the brain's endothelial cells, which line blood vessels, reproduces inside of them, and then moves on to invade the central nervous system.
“Crossing the blood-brain barrier is a rare event in part because this structure is designed to protect the brain from pathogens,” explained senior study author Christopher Hunter, Ph.D., chairman of the department of pathobiology at the University of Pennsylvania School of Veterinary Medicine. “And yet it happens, and we have now been able to visualize these events. It's something that no one had seen before.”
Over the years, several theories have been considered to explain how Toxoplasma could enter the brain. By elucidating the pathogen's path into the brain, the researchers hope to uncover treatment strategies that may be most effective in combatting the parasite before it wreaks its worst damage.
One invasion hypothesis has the parasite squeezing between the barrier cells while others think the parasite goes directly through a cell. A popular idea that is “beloved of microbiologists,” Dr. Hunter said, is the Trojan horse theory, in which the parasite hitches a ride across the barrier while hidden inside an infected host cell.
The Penn researchers used a multi-photon microscope, which allows them to peer deep into living tissues without damaging them, to try to witness the parasite's invasion in action. In these studies, they used mice that had been specially bred to express a green fluorescent protein in their endothelial cells. They then infected the mice with modified Toxoplasma that expressed a red fluorescent protein.
The findings from this study were published recently in Nature Microbiology through an article entitled “Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system.”
After a week, the investigators saw endothelial cells in the brain that were infected, as well as evidence that the parasites were reproducing inside those cells. Two weeks post-infection, they saw that parasites appeared in the brain tissue adjacent to the endothelial cells. Finally, they were able to visualize parasites bursting out of infected endothelial cells, thereby introducing the parasite into the brain.
To further illuminate the mechanism by which T. gondii infects and disseminates through the body, the researchers looked specifically at levels of free parasites, that is, parasites that had not already infected or become engulfed by a host cell. To their surprise, they saw that a significant portion, around a third of the mouse's total parasite load, existed as free parasites in the blood.
“I think we expected to see a small number of parasites outside cells because they have to come out to move from cell to cell,” noted lead study author Christoph Konradt, Ph.D., a post-doctoral researcher in Dr. Hunter’s laboratory. “But I don't think anyone had fully appreciated the sheer number of parasites that are free and able to infect other cells in the vasculature.”
However, the presence of free parasites was transient. By ten days post-infection, most mice had no free parasites in their blood.
“From a treatment perspective that means if a pregnant woman gets infected for the first time, there is a fairly short period of time when the parasite can cross the placenta and affect the fetus,” Dr. Hunter said. “That tells us that targeting these stages in the blood during this narrow window could be effective at preventing congenital transmission.”
As a final test to see whether parasites could directly access the brain from the blood, the researchers infected mice with a mixture of normal parasites and mutants that were unable to reproduce, each labeled in different colors. They then showed that only the normal, reproducing parasite made its way into the functional brain tissue.
“This shows that the parasite has to replicate in order to spread from the blood into other tissues,” Dr. Konradt remarked. “That could mean a drug that blocks replication could be effective at preventing dissemination.”