1. Bettina Arkhurst Arkhurst 1
Trogocytosis between Toxoplasma gondii and host cell membrane during
invasion
Abstract
The identification of all of the potential mechanisms that Toxoplasma gondii adopts during
invasion is a crucial component in understanding the vast success of this parasite. In this study,
we used light microscopy and the application of membrane specific dyes to observe the
interaction between Toxoplasma gondii’s membrane and human foreskin fibroblast host cells.
We used two types of Toxoplasma strains: the type I RH strain and the type II PRU strain to
view the parasite’s exchange of membrane proteins with its host. This exchange of cellular
membrane, known as trogocytosis, has primarily been observed in regard to lymphocytes. The
results of the recorded interactions show the possibility of Toxoplasma gondii possessing the
ability to carry out trogocytosis. Our findings show that not only does Toxoplasma secrete
proteins into the host during invasion, but the parasite also shows evidence of exchanging
membrane proteins with the host cell. These results may lead to a new perspective on the role of
Toxoplasma’s cell membrane during invasion as well as shed light on the possibility of other
microbes possessing the ability to carry out trogocytosis.

2. Arkhurst 2
Introduction
Toxoplasma gondii (T.gondii) is an obligate intracellular parasitic protozoan that causes the
disease toxoplasmosis [1]. There are three highly prevalent clonal lineages of Toxoplasma gondii
in the United States: the type I, type II and type III strains. The type I strain is the most virulent
of the three and the type II strain is the most commonplace of all strains in the United States [2].
With its capacity to infect virtually any nucleated cell, Toxoplasma gondii has infected about 15-
30% of the world’s human population, yet many immunocompetent humans with toxoplasmosis
are unaware of the infection due to the ability of the host’s immune system to keep the parasite
in its bradyzoite form [3, 4]. The parasite initially infects its host in its haploid, quickly
proliferating tachyzoite form. With the stress of the immune system, the parasite develops into
its latent form -- the bradyzoite form [4]. If immunocompromised patients acquire
toxoplasmosis, the disease has the potential to cause serious complications such as toxoplasmic
encephalitis and ocular toxoplasmosis and in severe cases, ultimately result in fatality [5].
During invasion, Toxoplasma has been observed to secrete proteins into the host cell in order to
allow for a more efficient invasion process [6, 7]. With much research on the secretion of rhoptry
proteins and organelles into the host during invasion, there is a paucity of research regarding
the workings of Toxoplasma’s membrane in the invasion process. Trogocytosis is the exchange
of cellular surface molecules, which has mostly been observed in regard to human lymphocytes
[8, 9]. This past year at the Albert Einstein College of Medicine, the parasite Trypanosoma
cruzi, known for causing Chagas disease, has been found to carry out trogocytosis [10, 11]. Like
Toxoplasma gondii, Trypanosmoma cruzi invades the cell through the use of a parasitophorous
vacuole [12, 13]. Much research has gone into the parasite’s secretion of organelles into its host
during invasion yet, there is a very finite amount of research that has been conducted regarding
the interaction between the parasite’s exterior membrane and that of its host.

3. Arkhurst 3
Methods
This technique was used to evaluate the exchange of membrane spots between the parasites and
host cells. We used two strains of Toxoplasma, RH (type I) and PRU (type II), to compare
results.
Cell and Parasite Preparation.
The human foreskin fibroblast (HFF) cells were obtained from the ATCC SCRC-1041.1 (HFF-1
IRR) lineage. The HFF cells were allowed to replicate without becoming too confluent in the
flasks. As the cells were passed into T25 Corning® flasks in preparation for infection, other HFF
cells (passed from the same sample) were put into wells to become more confluent for invasion
synchronization and observation. The flask of the RH strain was infected 1 day prior to
experimentation and the flask of PRU was infected 2 days prior to experimentation. Both types
of Toxoplasma went through the following methodology simultaneously but separately.
On the day of experimentation, 50% lysate was put into each infected flasks. A sterile cell
scraper was used to collect the remnants of the lysed cells and to disrupt any cells left intact. The
contents were put into a 50mL tube. The mixtures were passed through 20G, 23G and 27.5G
needles in a 10mL syringe to disrupt the remaining cells. This mixture was then passed through
a 15mL filter membrane and centrifuged at 400g for 15 minutes.
The parasites were then resuspended in 5mL of media and counted. For counting, 10 µl of the
parasite solution was diluted in 990 µl of PBS, and 10 µl was put into the heomcytometer for
manual counting. The parasites were then diluted to obtain a dilution of 1x108 . These parasites
were then placed into a 15mL conical polypropylene tube. The parasites were washed with 5mL
of media without serum and spun at 800g for 5 minutes. This centrifugation occurred three

4. Arkhurst 4
times for each type of Toxoplasma. To prepare the parasites for the dyeing process, all tubes
were then protected from light with aluminum foil.
Application of Fluorescent Dye.
For dyeing the parasitic membrane, components from the Sigma- Aldrich PKH26 Red
Fluorescent Cell Linker Kit as well as a modified protocol were utilized.
The parasites were gently resuspended into 1mL of Diluent C for 5 minutes. Simultaneously, 4µl
of dye was placed into 1mL of Diluent C. The parasites were rapidly added to the tube of dye for
gentle mixing. After 5 minutes of mixing, the reaction was stopped by the addition of 2mL of
fetal bovine serum to the solution. Each type of parasite group was then washed 3 times at room
temperature with cell media containing no serum. After each wash, a new tube was used. At the
last wash, we counted the reminiscent parasites.
The parasites were then resuspended in invasion media (2% serum) in preparation to sync cell
invasion.
Syncing of Invasion.
The parasites were put into each well at the proportion of about 106 parasites/200µl.
We synced the invasion by placing the infected well plate in the refrigerator for 15 minutes at
4°C. Then, placed the plates at 37°C in a water bath for the first time trials of invasion and then
in the incubator for later times. The groups were fixed according to their predetermined time
courses (2, 5, 10, 30 minutes) with 2% paraformaldehyde in PBS solution. The wells were
washed with PBS 3 times to remove any remaining contamination. The cell’s DNA was labeled
with the fluorescent blue DAPI in the mounting media. The slides were then mounted for
observation.

5. Arkhurst 5
Results
For the evaluation of trogocytosis, we used the Nikon® Diaphot inverted fluorescent microscope
to observe any evidence of trogocytosis. The parasite’s membrane is dyed fluorescent red and
the nucleus of the human foreskin fibroblast cells are dyed with fluorescent blue DAPI. We
looked for sharp red fluorescent spots from the parasite invading the cell to evaluate
trogocytosis.
The Controls
For controls, we used dyed RH strain Toxoplasma gondii that had been boiled for 5 minutes.
Figure 1.Image of dead RH strain (left) dyed with the Sigma-Aldrich PKH26 Red Fluorescent dye. Image of
control HFF cells (right) with nuclei dyed with DAPI. Both images were captured at the 30-minutes post-
infection point. These control images show the possible denaturation of the RH parasite after the dyeing
process.

6. Arkhurst 6
2-Minute Interval of Invasion
Figure 3. The 5-minute interval of invasion with the RH strain on the left and the PRU strain on the right.
The RH strain displays evidence of trogocytosis while the PRU strain appears to be denatured from the
dyeing process.
Figure 2. The 2-minute interval of invasion showing the RH strain on the left and the PRU strain on the
right. There appears to be no sign of trogocytosis in the RH strain of the parasite but the PRU strain appears
to be displaying possible trogocytosis.

7. Arkhurst 7
Figure 4. The 10-minute interval of invasion with the RH strain on the left and the PRU strain on
the right. The invading RH strain shows clear evidence of trogocytosis at this time interval while
the PRU strain shows no evidence of trogocytosis at this interval.
Figure 5. The 30-minute interval of invasion with the RH strain on the left and the PRU strain on the
right. The PRU strain shows evidence of trogocytosis.

8. Arkhurst 8
Discussion
We found evidence of trogocytosis during Toxoplasma invasion in both the RH and PRU strains
but this phenomenon was not apparent at all time intervals. There is an inconsistency when
viewing the parasite’s membrane particles.
There was evidence of trogocytosis as early as 2 minutes after invasion. As seen in Figure 2 the
RH strain did not show evidence of trogocytosis at this point in time, but the PRU portrays
potential trogocytosis with the existence of two red spots above the parasite. At the 5-minute
interval (Figure 3), both the RH and PRU strain show portray trogocytosis. The RH strain shows
two distinct molecules of membrane while the PRU strain seems more damaged in nature. At
the 10-minute interval, the RH strain shows evidence of trogocytosis with two visible portions of
membrane on either side of the invading parasite (Figure 4). The PRU strain in the 10-minute
interval shows no visible spots of membrane by the parasite. Figure 5 shows the RH strain of
Toxoplasma invading the host cell with no evidence of trogocytosis while there is evidence of
trogocytosis for the PRU strain in the 30-minute interval image.
The results show evidence of trogocytosis, but the inconsistencies suggest that trogocytosis may
not occur during Toxoplasma invasion at all points in time. We attempted to minimize error by
using sterile equipment and attempting to synchronize invasion by cooling down the parasites at
4°C but, as in all experimentation, we had sources of error that could have altered our results.
The potential sources of error in this experiment were: contamination of the specimen,
mislabeling, the inability to fully control invasion time, denaturing of the parasite as seen in
Figure 1 and the lack of trials for the experiment. The resulting possibility of trogocytosis
between Toxoplasma gondii and the host cell brings into question whether other microbes can
carry out the process as well.

9. Arkhurst 9
Conclusion
More experimentation is needed on this topic. There is evidence of trogocytosis in our results,
yet it is not consistent. Future research on this topic will allow for a clearer picture of
Toxoplasma invasion. Plans for future research include identifying the membrane molecules
that Toxoplasma uses to integrate into the host’s membrane, observation of the possibility of the
host cell providing membrane proteins and attempt to see possible signs of trogocytosis between
Toxoplasma gondii and other types of host cells.
Acknowledgements
Thank you to Dr. Louis Weiss, Tatiana Paredes Santos and the scientists of the Albert Einstein
College of Medicine for their unwavering support throughout the duration of this experiment. I
would like to thank my family for their unwavering support throughout my life. I would also like
to thank my ASR mentors as well as my fellow ASR peers for their continued support in my
scientific endeavors.

10. Arkhurst 10
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11. This article has not yet been published
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