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

insight in how a parasitology laboratory everyday research looks like and how are scientific projects

planed.

I would strongly recommend this internship for anyone who is interested to broadening their

knowledge and laboratory experience doing a summer research project about P. falciparum.