Unexpected step in malaria invasion
Imaging reveals parasite’s transmission path
Most malaria vaccines work only between 20 and 50 percent of the
time. Thus, scientists are eager to understand more about how the malaria parasites
infect their host. New imaging data has revealed a previously unrecognized step
on the parasites’ transmission path, which could aid in the development
of better vaccination strategies.
Mouse malaria parasites (green) glide in circles on a glass surface (red) because they
have a curved shape.
Malaria is transmitted by an infected mosquito
into the skin of a mammal and travels to the liver to infect red blood cells and
cause disease. Friedrich Frischknecht from the department of parasitology at the
University of Heidelberg Medical School in Germany and his colleagues, working at
that time at Pasteur Institute in Paris, have studied how the malaria parasites
move once inside the host. Using wide-field imaging and confocal microscopy, they
found that parasites seem to experience various fates inside their mammalian hosts.
Using wide-field imaging and confocal microscopy,
researchers have discovered that parasites move in various paths in their host.
Each color represents a different parasite’s path inside the skin of
a living mouse. Images courtesy of Sylvia Münter, University of Heidelberg
As reported in the February issue of
Nature Medicine, the researchers let a mosquito infected with green fluorescent
clones of the mouse malaria parasite Plasmodium berghei bite the ear of a
hairless mouse. They observed the site using a Zeiss or an Olympus inverted microscope
and found that once the mosquito bites, it distributes the parasites over an area
of about 400 x 600 μm. The parasites in the skin displayed a forward-gliding
motion, which seemed like a random path. “The parasites move in a peculiar
way because they are shaped in a slightly curved manner,” Frischknecht said.
He explained that, in vitro, the parasites always move forward in a circle because
they are curved like bananas, but that, inside the skin, they appear to move randomly,
probably because of all the dense tissue.
The researchers initially captured
video images of the parasites’ journey using an imaging system from Till Photonics
GmbH of Gräfelfing, Germany, and later upgraded to a spinning disk confocal
microscope from PerkinElmer Inc. of Wellesley, Mass. They tracked the blood vessels
using either transmission light microscopy or by injecting red fluorescent bovine
serum albumin into them. As expected, some parasites invaded the blood vessels,
usually within the first 20 minutes after the bite. They glided into the skin and
attached to the blood vessel walls, which they penetrated until they were suddenly
carried away at the same speed of the circulating red blood cells, indicating their
invasion into the vessel.
To determine just how many of the parasites
were actually invading the blood vessels, the scientists, in 12 experiments, counted
about 20 parasites on average in the mouse’s skin just after the mosquito
bite. They also observed that some didn’t move in a forward motion but instead
drifted in a sideward and low-velocity movement. They carefully watched more of
the video imaging and realized that the parasites were drifting because they were
in the lymphatic vessels.
They confirmed this using confocal
microscopy and by injecting more parasites with red fluorescent dextran. The images
showed that, after about an hour, half of the parasites left the skin site and about
70 percent of them invaded the blood vessels, while 30 percent invaded the lymphatic
vessels. They were surprised by these results.
The researchers then changed the model
system from the ear to the footpad of the mouse — where it is easier to extract
the draining lymph nodes — to further analyze the fate of the parasites that
entered the lymphatic system. After allowing 10 infected mosquitoes to bite the
footpad, they dissected the two draining lymph nodes and found about 50 parasites
inside the first lymph node, which represented about 25 percent of the injected
parasites, but found none in the second.
To assess the fate of the parasites
in the lymph node, they labeled them with antibodies and performed time-lapse imaging
using a spinning disk confocal microscope with a Hamamatsu CCD camera. Image acquisition
time varied between 20 ms and 2 s, depending on the tissue depth, and they recorded
images for up to 25 minutes at a time. Image files were processed using the National
Institutes of Health’s ImageJ and Adobe software. They found that, after one
hour, most of the parasites still had their characteristic “banana”
shape, but that after four hours, they appeared damaged, and at eight hours, most
of them had lost their fluorescence.
The researchers then compared how parasites
develop in the lymph node as opposed to how they develop in the liver (the final
stop for those that traveled through blood vessels). After about 15 hours, a small
proportion of the lymph node parasites appeared the same as those developing in
the liver. But after 24 hours, the liver parasites had grown much more than the
ones in the lymph nodes. And after 48 hours, the researchers could no longer detect
any parasites in the lymphatic vessels.
“We don’t know whether
they are eaten up or whether they deteriorate — or what happens to
them,” Frischknecht said. But he stressed that just knowing that some of them
enter the lymphatic vessels shows that the parasites seem to meet various fates
in their host, which could be important for future vaccination development.
The researchers would like to understand
more about the parasite’s movement and outcome inside mammals. They would
like to combine light microscopy with cryoelectron tomography to observe them more
closely. Frischknecht said that they will image the parasites using a light microscope
and then will freeze them and look at them with the cryoelectron microscope to
see if they can learn more about their three-dimensional structures implicated in
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