Abschlussbericht DAAD RISE Weltweit 2016
''Quinine and chloroquine induced hemozoincrystals in Plasmodium faciparum''
an der McGill University
vonMilica Moskovljevic
08.08.2016-03.10.2016
Land: CanadaName: Milica MoskovljevicSupervisor: Petra RohrbachInstitute: Institute of Parasitology McGill UniversityInternship period: 08.08.2016-03.10.2016
In the summer of 2016 as a DAAD scholar and thanks to RISE Weltweit Program I was able to do a
research internship at McGill University Institute of Parasitology. At the time of application I was
enrolled in fifth semester of pharmacy studies at Faculty of Biosciences Heidelberg University.
During my university courses I became interested in antimalarial therapy and the challenges that it
faces as well as investigating the mechanisms that cause resistance. For that reason I applied for a
research internship at Prof Rohrbach's laboratory which focuses on malaria parasite- Plasmodium
falciparum.
General part
1. Travel preparation
1.1 Visa application
As soon as I got a conformation e-mail from DAAD in February about being successfully
matched for the research internship program that I applied for, I contacted my future supervisor and
determined the duration of my internship.
I also started to prepare the documents required for visa application. Visa application process
is conducted online by CIC (Citizenship and Immigration Canada) and the most current information
can be found on their website http://www.cic.gc.ca/english/index.asp. It is important to always
check the most recent document requirements for visa application cause Canadian government tend
to change them from time to time.
Generally, before applying for visa it is required to make an account on CIC website and answer
some question related to your stay in Canada. For us, the DAAD scholarship holders, the suitable
visa option is “Work permit with Labour Market assessment exemption“ (LMIA exempt). But the
application process itself is not really appropriately adjusted and there are some document that
cannot be displayed like "Proof that you meet the requirements of the job being offered" or
"Employment records". However, it necessary to upload DAAD-Zusage, an invitation letter from
your supervisor, your certificate of enrollment at home university and possibly the funding
certificate of the DAAD. It is needed to fill out a work permit application form (IMM1295E) and a
family information form (IMM5645E). You will also be asked to upload further documents such as
the scan of your passport, a photo (in appropriate PDI format), your CV, transaction of payment
form you supervisor etc. It is always the best to check the document requirements for that certain
year on CIC website.
Earlier the form "IMM5802 Offer of Employment to a Foreign National LMIA Exempt ", which
should be filled by the supervisor ,was needed. Citizenship & Immigration Canada has replaced the
form IMM5802 with an online process on a portal (again need to be filled out by your supervisor).
For this part I have been in a permanent contact with my supervisor via e-mail and she got the most
recent information from McGill University administration office. The supervisor needs to pay a
230$ fee for your visa application.
In order to fill the your work permit application form (IMM1295) correctly you should also know
the LMIA exemption title, code and job title. This information you should be able to get from from
your supervisor.
After uploading all the necessary documents and paying the fee (visa fee is 155 $) you will
receive a conformation from CIC via your account that they got your application. The time they
needed to process my application was really short- about 1 week.
Also all further information for the undergraduate research trainee at McGill university can be
found on this link http://www.mcgill.ca/students/records/trainees.
1.2 Finding an accommodation
Finding a housing at university campus is very difficult given that German's school year
differs from Canada's. Fall semester at McGill University starts in September and dorm rooms have
to be free by August 15th for the regular students. Therefore the only option is looking for a private
housing.
McGill University has two campuses, both situated on the Island of Montreal. The Institute of
Parasitology is located at the Macdonald campus in Sainte-Anne-de-Bellevue, the western tip of
Montreal island, 30 km away from downtown.
Firstly, you can always search for an accommodation in a Facebook group for international student
looking for a housing in Montreal. Here the student post different room offers almost everyday, but
the only thing to be aware of is that you would want to avoid any lease signing cause you will be
staying in Montreal just for a few months. Secondly, the websites where you can look for
housing/accommodation in Montreal are airbnb, craigslist and kijiji. I decided to also contact
students from McGill university who were as well DAAD scholarship holders for RISE Germany
Programe via 'RISE get connected' (DAAD will send you an e-mail with all RISE scholarship
holders contact data). Some of them were very kind to offer me some advice and help me get into
MacDonald campus Facebook group.
Finally I opted for a room in downtown for which I found an ad on kijiji website and I
contacted the landlord via e-mail. The connections between Montreal downtown and the institute
are the express public buses. The ride usually took one hour, but this varied depending on the time
of day. During rush hour the ride was longer and especially during my stay in Montreal all major
highways were under reconstruction. The reason for that is the fact that in 2017 it will be the 375 th
anniversary of Montreal. So, if you choose to rent a room somewhere downtown, be prepared to
commute daily for about 3 hours .
On the other hand if you choose to stay somewhere near to MacDonald Campus, you should know
that Sainte-Anne-de-Bellevue is a very small town with lots of green areas and beautiful family
houses situated directly on Sainte-Anne-de-Bellevue Canal. This canal set of locks linking Lake
Saint-Louis and Lake of Two Mountains. If you need larger supermarkets or a certain bank, you
would also need to take a bus.
1.3 Life in Montreal
Montreal is the largest city of province Quebec and the second largest city in Canada.
French is an official language, but McGill University is an English-speaking university and you
would most often hear English around the campus. West part of Montreal Island is mainly
anglophone. Being able to speak the French language is definitely an asset in Montreal, but the most
of people can speak some English, so it is not an issue to speak just English (especially younger
population is mostly bilingual).
Montreal has a 375 year long history and the European influence can be observed in cities
architecture. Its unique combination of European and North American influence and dual French-
English mix adds dynamism to the city. Montreal has over 250 theaters and 90 festivals and it also
offers great variety of international cuisine.
The temperature in the city in August is often above 30 ºC that is why during the summer
Montreal offers great variety of music festivals and other cultural activities outdoors. Every Sunday
in Park Jean-Drapeau there is a Picnic electronique, an electro music festival with local DJs.
Montreal doesn't lack in parks and green surfaces where you can hike, go for a run, or even have a
barbecue. Mont Royal is a mountain in the heart of the city after which Montreal got its name. If
you go to the top of the mountain, there is a spectacular panorama view of the city.
Personally, the most interesting part of the city is Sherbrooke. It is filled with local groceries, art
and fashion ateliers, bakeries and restaurants. In Sherbrooke you can really experience the essence
of Quebec culture and try typical local food such as poutine and smoked-meat sandwich.
I would also recommend to pay a visit to Jean Talon farmers market and try fresh local fruit and
vegetables. I would definitely suggest going to Botanical Garden and Insectarium (they have some
awesome bugs and butterfly collections there) and with your McGill student ID you will get a
discount on the entrance price.
1.4 Travel in Canada
For trips within Canada especially Quebec and Ontario traveling with megabus is your
cheapest and best option (https://ca.megabus.com/landingcanada.aspx?return=/default.aspx). The
sooner you book your ticket the cheaper it will be. This bus company is something similar to
flixbus/meinfernbus here in Germany. I traveled with megabus from Montreal to Toronto over the
weekend and I can say that the trip was more that fine. It took about 6 hours to get to Toronto and it
might be even faster than traveling by train.
There is also a possibility to travel to the US cities on the east coast that are not that far away from
Montreal. A traveling agency called interstude organized different trips for international students in
Montreal. I haven't traveled with them, but all the information are available on their website.
Laboratory part
2.1. Research internship
Prof Rohrbach's research group focuses on studying malaria parasite physiology using live
cell imaging and fluorescence microscopy in order to achieve a better understanding of the function
of specific parasite organelles and their role in antimalarial drug resistance.
Initially my supervisor suggested two projects that I was about to conduct during my stay in the
laboratory.
First one included isolation of protein pHluorin, a pH-sensitive green fluorescent protein, and
transfection into Plasmodium falciparum in order to measure more accurate the pH value in
parasite's cytosol and organelles.
In the second project I observed the changes in parasites' morphology of resistant and sensitive
strains after treating them with different antimalarial by using live cell imaging technique. Here I
was especially interested in hemozoin crystals compartments appearing outside of parasite's
digestive vacoule.
The blood bank made a shipment error and delivered the a yearly amount of blood to the laboratory
back in spring. That was the reason why the laboratory was out of blood for the whole summer and
I wasn't able to cultivate the parasites for the first three weeks of my internship.
2.2. Introduction
Malaria is one of the most common infectious diseases in the world caused by the protozoan
parasite Plasmodium. Plasmodium falciparum, which causes the most severe form of malaria,
replicates within red blood cells. It is the most virulent and its pathology is characterized by severe
anemia or the development of cerebral malaria, generally leading to death if left untreated.
Health impacts of malaria are enormous, causing disease in over 500 million individuals and
resulting in the death of approximately one million young children each year. Although extensive
malaria control measures have significantly decreased the incidence of malaria worldwide,
antimalarial drug resistance remains a serious concern and threat.
Infection is introduced to humans in the bite of an infected female Anopheles mosquito.
Once an erythrocyte has been invaded, the parasite feeds on the hemoglobin, and this stage is
known as a trophozoite. During its growth within the red blood cell, the parasite degrades
hemoglobin within a digestive vacuole (DV) to supply amino acids, releasing the toxic product
heme- ferriprotoporphyrin IX. While free heme is detoxified by enzymatic degradation in
mammals, the malaria parasite has evolved a distinct mechanism for heme detoxification. During
detoxification, free heme is converted into a crystalline form called hemozoin (Hz). The hemozoin
appears as characteristic brown crystals in the digestive vacuole of the parasite.
Inhibition of Hz formation is one of the most important factors targeting the malaria parasite
because the process of Hz formation is important for the survival of Plasmodium species. Several
antimalarial drugs are thought to inhibit Hz production. However, resistance to currently used
antimalarial drugs has led to an urgent need to develop new and effective drugs. Quinoline
antimalarials, such as chloroquine (CQ) are known to bind to multiple precrystalline forms of heme
in different ways and thus presumably inhibit Hz-crystal growth by sequestration of monomeric
and/or dimeric heme. Disruption of the DV membrane and leakage of DV contents into the cytosol
have been hypothesized to be the main contributors to parasite death after CQ exposure. Free
cytotoxic heme accumulates in the parasites, causing their deaths. The mechanism of action of
quinine (QN) has not been fully resolved. The most widely accepted hypothesis of its action is
based on the well-studied and closely related quinoline drug, chloroquine.
Previous studies showed that verapamil is a chemosensitizer of CQ-resistant parasites. Therefore,
the effect of verapamil on the formation of cytosolic Hz-compartments in CQ-resistant parasites
was investigated.
The aim of my project was to observed the appearance of dark-stained Hz-containing
compartment in parasite cytosol over different time points in sensitive and resistant strains induced
after treating them with antimalarials using live cell imaging.
2.3 Methods
In the first project I was supposed to transfect Plasmodium parasites using integrase-
mediated attB × attP recombination. Because of the low transfection and recombination efficiencies
in P. falciparum, I firstly transformed E. coli XL1-Blue competent cells with pGEX-2T vector
which contained gen for pHluorin in order to get the higher yield. The isolation of plasmid DNA
from transformed bacterial cells was conducted by Miniprep kit. This isolated plasmid DNA is
supposed to be cut on certain enzyme cute sites, so that pHfluorin gen could be now integrated in
pLN- PfCRTs. Such pLN- PfCRT vector that carries pHfluorin gen s an insert is supposed to be
transfected in P. falciparum. Unfortunately, after trying to optimize the Miniprep kit, the isolation of
pGEX-2T DNA didn't work and therefor I wasn't able to conduct this project further.
I started the second project later because of the blood shortage in August. This project
included live cell imaging with a confocal microscope. Here two P. falciparum strains were used: a
CQ sensitive one- 3D7 and a CQ resistant one- Dd2. Two antimalarial drugs were used chloroquine
and quinine. After treating the parasites with the antimalarials I would monitor the Hz-crystals
compartments appearing outside of their digestive vacoule in trophozoite stage parasites for 4h.
Cultivation of Plasmodium falciparum
In order to start my project with Plasmodium parasites I needed to learn how to cultivate
them. Before cultivation parasites need to be thawed and blood washed in order to be left just with
erythrocytes.
For the optimal growth of parasite, the cultures needed to be taken care of i.e. the fresh media RPMI
1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, gentamicin (20 µg/ml) 100 µM
hypoxanthine, and 0.5% AlbuMAX I is to add every day. In order for cultures not to be stressed the
prasitemia also needs to be kept low and therefor I needed to learn how to dilute the cultures.
In their growth cycle there are few stages that can be differed which can be distinguished observing
the culture under the light microscope. For cultivation I needed parasites to be in trophosoite stages
and I also learned how to synchronize the cultures.
Parasites were maintained at 37°C under an atmosphere of 5% CO2, 3% O2 and 92% N2.
2.4 Results
Live cell imaging reveals that Hz crystals move freely within the DV in a disorganized manner.
After treating the 3d7 parasites with CQ and QN Hz crystals were detected in cytosolic
compartments outside of the DV (Figure 1. and 2.).
Figure 1. Untreated 3D7 trophozoite (left) and 3D7 trophozoite after 45 min incubation with CQ (right); DV-digestive vacoule, RBC-red blood cell, Hz-compartments are marked with red arrows.
It was also observed that Hz-compartment appeared sooner when 3D7 strain was treated with CQ
(Figure 2. and 3.). First Hz-compartments in the cytosol were observed after 45 min after treatment
with CQ, whereas in the case of QN it took 2h for first Hz-compartments to appear outside of the
DV.
It was observed that Hz crystals appeared smaller and move much more rapidly in Dd2 CQ-
resistant strains. Dark-stained Hz-containing appeared in the parasite cytosol after 5 h incubation
with CQ and verapamil (Figure 4.) .
Figure 2. 3D7 parasites treated with quinine after 2h (left) and 3h (right); Hz-compartments outside of theparasite DV are marked with red arrows.
Figure 3. 3D7 trophozoite after 45 min(right) and after 1 h (right) incubation with CQ ; DV- digestive vacoule,
Hz-compartments are marked with red arrows.
Figure 4. Dd2 trophozoite incubated with CQ and verapamil after 5 h ; DV- digestive vacoule, Hz-compartmentsare marked with red arrows.
Conclussion
Finally, I can summaries that I have learned various aspects of confocal microscopy and live cell
imaging method. I have also expended my knowledge in some molecular methods and learned how
to cultivate malarial parasites nad take care of the cultures. Also, I have gained a very thorough