Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

Parasitology Section
Survival strategies of
Plasmodium parasites in hepatocytes
Zusammenfassung
Wir benutzen den Nagerparasiten Plasmodium berghei
als einen Modellorganismus, um die Überlebensstrategien
von Plasmodien in Hepatozyten zu untersuchen.
Im Hepatozyten leben die Plasmodien
zunächst in einer parasitophoren Vakuole. Während
der Merozoitenbildung wird die Vakuolenmembran
zerstört und die Parasiten gelangen ins Wirtszellzytoplasma.
In vitro konnten wir beobachten, dass sich
die infizierten Zellen nach der Auflösung der Vakuolenmembran
abrundeten und frei im Medium
schwammen. Diese frei schwimmenden Zellen zeigten
intrazellulär Eigenschaften von apoptotischen
Zellen. Die Zellmembran blieb dagegen zunächst intakt,
was den Schluss nahe legt, dass infizierte Zellen
nicht von phagozytierenden Zellen erkannt und
eliminiert werden können. Untersuchungen der molekularen
Mechanismen dieser Parasit-Wirtszell Interaktionen
sollen helfen, die Biologie des Leberstadiums
von Plasmodien besser zu verstehen und Strategien
zu entwickeln, die die Entwicklung der Parasiten
in der Leber hemmen.
Summary
Using the rodent parasite Plasmodium berghei as a
model system we investigate survival strategies of Plasmodium
parasites in hepatocytes. Within the host cell
Plasmodium parasites live initially in a parasitophorous
vacuole. During merozoite formation the vacuole membrane
is destructed and the parasites lie free in the host
cell cytoplasm. In vitro we observed that upon the degeneration
of the PVM, parasitised host cells rounded
off and floated in the culture medium. These floating
cells exhibited intracellular features of apoptotic cells.
However, at first the cell membrane of the host cell remained
intact suggesting that infected cells cannot be
recognized and eliminated by phagocytising cells. Investigating
the molecular mechanisms of these parasite-host
cell interactions will help to understand the biology
of the liver stage of Plasmodium and to improve
strategies to inhibit the development of the parasite in
the liver.
Project Description and Results
Malaria is caused by parasites of the genus Plasmodium
and is transmitted by Anopheles mosquitoes. During
a blood meal infected mosquitoes inject Plasmodium
sporozoites into the skin of the mammalian host.
From there the sporozoites reach the circulatory system
and are transported to the liver, where they infect hepatocytes.
The invasion of hepatocytes by Plasmodium
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sporozoites is a well-documented event, but little is
known how the parasite survives in the host cell and
how exoerythrocytic merozoites are finally liberated.
Although the host cell has principally the capacity to
counteract the infection by committing suicide, Plasmodium
parasites can obviously grow and develop intracellularly
suggesting that the parasite interferes with
pathways leading to programmed cell death (apoptosis).
In order to investigate whether parasite infection protects
the host cell from apoptosis, we treated two days
infected cultures with concentrations of peroxide which
induces programmed cell death in non-infected cells.
Immunofluorescence studies revealed that infected
cells are indeed protected against peroxide treatment
confirming our assumption that the presence of the parasite
confers resistance to apoptosis to its host cell.
As long as the parasite develops and grows in the hepatocyte
it is an advantage for the pathogen to support
the survival of the host cell. But what happens when
merozoites are formed which must leave the cell to infect
red blood cells? We examined the final stage of
parasite development in transformed hepatic cell lines
and in primary hepatocytes in vitro and observed very
interesting changes in infected cells. Three days post
infection most P. berghei-infected cells lost adherence
to the surface and to other cells in the culture. They
rounded off and floated into the culture supernatant.
Floating infected cells exhibited features of early apoptotic
cells including nuclear condensation of the host
cell nucleus, cytochrome c release from the mitochondria
and caspase activation. However, another early
marker of apoptosis, the loss of cell membrane asymmetry,
could not be detected for several hours in infected
floating cells and was only apparent briefly before
merozoites are released.
What could be the biological relevance of these observations?
Our working hypothesis is that in vivo parasitised
cells loose contact to their neighbouring cells
and are bulged out between the normal hepatocytes.
The later phases of intracellular parasite development
are taking place in the space of disse or in the sinusoids
of the liver (figure 1). Since the membrane of infected
floating cells was found intact it is reasonable to
assume that parasitised cells cannot be recognized by
phagocytosing cells of the immune system. We believe
that the observed membrane integrity in an otherwise
apoptotic cell is actively preserved by the parasite and
can thus be considered as a so far unknown immune
evasion mechanism.