1. Rhomboid Protease 3 (ROM3) is Necessary for Plasmodium Yoelii Transmission
Abstract
Malaria is a serious disease caused by an
apicomplexan parasite transmitted to humans
by a mosquito vector of the genus Anopheles.
Each year, approximately 1,500 cases of malaria
are reported in the United States. On a broader
scale, the World Health Organization estimates
that malaria caused 219 million clinical
episodes, and 660,000 deaths in 2010. In fact,
3.3 billion people live in areas at risk of malaria
transmission. Efforts are being made by
scientists to better understand the disease and
how to prevent its transmission. It was
discovered while examining the Plasmodium
Yoelii rodent model, that when the Rhomboid
Protease 3 protein (ROM3) is knocked out, the
parasite is unable to complete the mosquito
cycle and therefore cannot be transmitted back
to its host by the mosquito vector. What we
chose to focus on in our experiment is in what
stage the ROM3 protein is present, and how the
lifecycle is affected when the protein is knocked
out.
Experiment Background
For our experiment, we tested both the
malaria wild type (no mutations) and the
Rhomboid Protease 3 Knock Out (ROM3 is
mutated). We infected mice with either the wild
type parasite, or the ROM3KO parasite, and
found that all the mice display the same
phenotype. By taking blood samples from the
mice at each stage, we found that all blood
stages exhibited the same morphology. The
Wild Type and ROM3KO parasite were equally
as deadly to the mice once the mice were
infected. Although, there is a block in the
ROM3KO transmission from the mosquito to its
host, preventing the mouse from getting
infected. To find where the mutation occurred,
we took samples from both the wild type and
the ROM3KO at each stage of the lifecycle. In
doing this, we found that ROM3 was present in
two stages, the Oocyst and Gametocyte stage.
ROM3 protein
was identified in
the gametocytes.
Malaria Lifecycle
http://www.cdc.gov/malaria/about/biology/
Sarah Simon, Catherine de Beaumont PhD, Li-Min Ting PhD, Kami Kim M.D, PhD
Department of Medicine (infectious diseases), Microbiology & Immunology, Pathology
Oocyst Stage
To test whether or not the lack of transmission
was due to a decrease of oocyst numbers in the KO
parasite, we looked at the mosquito midgut. We
counted the number of oocysts for both the wild
type and the ROM3KO at day 6 to day 8 post
mosquito infection after a stain in 0.1%
mercurochrome. We concluded that there is no
difference in the number of oocysts. Therefore, we
could determine that the lack of transmission is
not due to a decrease in oocyst numbers in the
ROM3KO parasite.
We noted that the ROM3KO oocysts were empty,
whereas the wild type oocysts were organized with
forming sporozoites.
Plasmodium Yoelii Gametocytes
What Prevents Transmission in the ROM3KO Parasite?
Malaria is transmitted through the release and
injection of sporozoites to its host. What was noted
previously in our experiment, was that the cycle that
occurs in the mosquito presents a block: in the ROM3KO
line, sporogony (formation of sporozoites in the oocyst)
does not occur. When observing the wild type parasite,
we counted over 1,000 sporozoites in each oocyst. In the
ROM3KO parasite, the oocyst remains empty and no
sporozoites form, thus, if mice are bitten by mosquitos at
the end of the mosquito cycle, wild type parasites will
infect the mice but the KO parasite will not.
These photos were obtained using a
confocal microscope and prepared by
performing immunofluorescent assays
on thin blood smears from a mouse
during the gametocyte stage.
In observing these photos, we were able
to conclude that anti Plasmodium Yoelii
ROM3 antibodies were raised in mice,
and stained Plasmodium Yoelii
gametocyte enrichment in
Immunofluorescent Assay. Moreover, the
antibodies cross-reacted against
Plasmodium falciparum gametocytes (the
deadliest of the 5 parasites that cause
malaria in humans).
Plasmodium Falciparum Gametocytes
Method
Gametocytes were enriched from
Plasmodium Yoelii infected blood
from a mouse by a density gradient,
or from a Plasmodium Falciparum
culture. The samples were then
smeared on a slide, fixed in
methanol: acetone (1:1), and
incubated with the mouse anti-ROM3
antibody. An anti-mouse coupled to
biotin was then probed, and finally a
fluorophore coupled to Streptavidin
was incubated. DAPI was added in
one of the final washes that stains
cell nuclei. The slides were then
examined under a confocal
microscope.
Acknowledgments
Albert Einstein College of Medicine, Montefiore University Hospital, High School
Summer Research Program