Anopheles:The Half Blood Princess
Team IISER Pune India 2020
Team:IISER-Pune-India/Poster
Background and Inspiration
Background
Malaria is a mosquito-borne, life threatening disease caused by the Plasmodium parasite. In 2018 alone, it was responsible for 228 million infections and 405,000 deaths. Out of the 5 species of these parasites that cause malaria in humans, Plasmodium falciparum is the deadliest[1]. A malarial parasite completes it lifecycle is two hosts, humans and female Anopheles mosquitoes.Over the past few decades it has been observed that plasmodium has gained resistance against commonly used drugs. Resistance occurs naturally but with the excessive use of antibiotics it has increased significantly. Currently, artemisinin is the most potent anti-malarial drug and it has been used in Artemisinin Combination Therapy (ACT). However, there is growing evidence that the parasite is gaining resistance against artemisinin worldwide.
Inspiration
When brainstorming different project ideas, we decided to pick a problem that was affecting the communities around us. Based on our experiences with mosquitoes in our college, out of curiosity we looked up mosquito borne diseases. The statistics we found for malaria were shocking. We looked up various interactions that were crucial for the entry of the parasite into RBCs, and hence decided to come up with a drug library which will be helpful in case of solving the problem of resistance.To further expand our project, we decided to graft our inhibitory sequence into something so that it can be administered as a drug. And that’s when a team member came across the work of Team Heidelberg 2014 [2]. We then decided to come up with a cyclotide based drug which can be easily administered and is stable.
Human Practices and Social Outreach
Experts:
Dr. Gamboa (malarial scientist): Accessibility issues of rural populations → developed an easy-to use, cheap, diagnostic kit
Dr. Velavan (malarial scientist): P. falciparum causes the deadliest form of Malaria → focused on P. falciparum instead of P. vivax
Dr Priyanka Devgun (community medicine doctor): Attitudes toward Malaria are lax → Designed an effective public engagement strategy
Dr. David Norman (structural biologist): Provided input and feedback with structural modeling of cyclotides
Poornima Raveendran (Senior Teaching Associate): Accessibility issues with the diagnostic kit → aimed to create instructional videos in vernacular languages and further develop software to detect Malaria without preprocessing images.
Sriram Raghavendran (Joint Executive Director, Star Health and Allied Insurance): Diagnostic kit must be as simple as possible → decided to split the kit into two components, Reusable and Consumables and test whether pre-assembling the foldscope before distributing it would be possible
Feedback from doctors:
Dr. Nilofar Bijali (worked in a public health organization in a tribal area): Tests needed to be conducted to see if our diagnostics kit was better than Rapid Diagnostic Tests (RDTs)
Dr. Rituja Sardesai (previously worked in a rural primary health center): Our instruction manual for the diagnostic kit was well-made and our instructional video for the foldscope required translation into vernacular videos.
Dr Akshay Wagh (currently works at a rural primary health center): It would be necessary to perform field studies and efficiency studies before continuing the development of such a kit.
Dr. Roshani Rameshwar Sagane (currently works at a rural primary health center): Maximum pictorial representation is necessary in the instruction manual that we have developed. She also had suggestions on the distribution of our diagnostic kit.
Public Engagement:
Webinar (via collaboration): A webinar for school students, highlighting the dangers of mosquito-borne diseases, and what we could do to combat them.Radio show: An interview on a radio channel, where a team member spoke about the deadliness of Malaria and how good hygiene can prevent the spread of the disease.
Journal publication (via collaboration): The iGEM Vector and Muggle journals, created by the MSP-Maastricht team, contain peer-reviewed articles from iGEM teams from across the globe. We submitted an article and were part of the peer-review process.
Malaria song: In order to spread awareness about the seriousness of Malaria and how it could be combated, our team wrote, composed, produced, and sang a Malaria song, which can be found on our wiki!
Tangram activity: We developed a module to teach high school students about synthetic biology. This used tangrams to teach kids the basics of genetic engineering.
Experimental Design
Gene circuits
Below are some of experiments devised by us to perform in the lab when possible.
Ni-NTA solid phase protein Binding assay:
This assay has been designed based on the facts that proteins with Histidine tags can be selectively immobilized using a chelating agent such as Ni-NTA (Nickel Nitriloacetic acid). The interacting proteins bind to each other and the concentration of the bound protein can be found, enabling us to measure the amount of host protein bound to the parasite protein both in the presence and absence of our inhibitory drug.
Half maximal inhibitory Concentration assay (IC50):
This assay is used to measure the concentration of the proposed drug that is required to inhibit a particular process or molecule by 50 percent. It uses a fluorescent DNA binding dye SYBR Green1 to measure the DNA content of the parasite present in the sample.
RBC Invasion assay:
This is done by counting the number of parasite infected RBCs by flow cytometry in the sample. It exploits the fact that parasites have DNA but not RBCs and can be differentially stained by DNA binding dyes.
Below are some of experiments devised by us to perform in the lab when possible.
Ni-NTA solid phase protein Binding assay:
This assay has been designed based on the facts that proteins with Histidine tags can be selectively immobilized using a chelating agent such as Ni-NTA (Nickel Nitriloacetic acid). The interacting proteins bind to each other and the concentration of the bound protein can be found, enabling us to measure the amount of host protein bound to the parasite protein both in the presence and absence of our inhibitory drug.
Half maximal inhibitory Concentration assay (IC50):
This assay is used to measure the concentration of the proposed drug that is required to inhibit a particular process or molecule by 50 percent. It uses a fluorescent DNA binding dye SYBR Green1 to measure the DNA content of the parasite present in the sample.
RBC Invasion assay:
This is done by counting the number of parasite infected RBCs by flow cytometry in the sample. It exploits the fact that parasites have DNA but not RBCs and can be differentially stained by DNA binding dyes.
Modeling
- Five interactions of PfEMP1 and PfRH5 proteins with various human proteins were studied. These interactions are crucial for erythrocyte invasion and for the survival of Plasmodium falciparum. [3] ,[4],[5],[6],[7]
- Peptides were designed by saturation mutagenesis and scoring parts of human proteins.
- Interaction energies scores of mutant peptides with Plasmodium proteins were determined.
Below are peptide protein pictures of 2 interactions
- Best scored peptides chosen for MD simulations. The abundance of hydrogen bonds between the inhibitor and the protein along with the time evolution of distance between them in the triplicate MD runs were analysed to study their stability.
- Grafting of these inhibitor peptides onto the 6th loop of modified kalata B1.
- Molecular dynamic simulations were performed on these grafted cyclotides for studying the stability.
14 potential peptide inhibitors were designed against the five identified Plasmodium- human protein protein interactions.
Advanced modelling studies are needed for studying the structure and stability of the grafted cyclotides in silico.
DeLeMa Detect (DEep LEarning for MAlaria Detection)
DeleMa-Detect is a Web App that can batch-process and classify blood smear images as malaria infected or not within a few seconds. A deep learning model built by rigorous training on a large dataset (Rajaraman et al. 2018) performs the preprocessing and classification at the back end. The model works on transfer learning and is built on top of the Mobilenet_v2 convolutional neural network built by Google AI, primarily for mobile phones, and achieves a validation accuracy of ~ 96%.
The foldscope is an origami-based microscope that is cheap, lightweight, and small(70 x20 x 2 mm3). It does not require an external power source and allows for a wide range of magnification by changing the spherical ball lens, as magnification varies inversely with ball-lens diameter. [8]
The paper centrifuge is a centrifuge that is low cost, lightweight, and human-powered. It can achieve the required centrifugal force to separate the layers of blood [9] and together with the foldscope, can enable us to obtain a blood smear image with ease.
The user manual created by our team can potentially aid medical professionals to operate the foldscope and paper centrifuge effectively.
The foldscope is an origami-based microscope that is cheap, lightweight, and small(70 x20 x 2 mm3). It does not require an external power source and allows for a wide range of magnification by changing the spherical ball lens, as magnification varies inversely with ball-lens diameter. [8]
The paper centrifuge is a centrifuge that is low cost, lightweight, and human-powered. It can achieve the required centrifugal force to separate the layers of blood [9] and together with the foldscope, can enable us to obtain a blood smear image with ease.
The user manual created by our team can potentially aid medical professionals to operate the foldscope and paper centrifuge effectively.
Description
Therapeutics:
We aim to create a library of orally administrable peptide drugs which target multiple, crucial host-pathogen interactions in the blood stage of Plasmodium falciparum malaria, effectively preventing the parasites from infecting human red blood cells (RBCs) and effectively circumventing the problem of drug resistance by using a different drug from the library when the parasite gains resistance to a particular drug.The peptide drug consists of two parts: An inhibitory peptide and a cyclotide* backbone (Kalata B1).
*Cyclotides are plant-derived circular peptides which are stable to degradation, highly tolerant of sequence variability, orally bioavailable and have been used previously to design drugs. We will use it as a drug scaffold.
Diagnostics:
The current gold standard for the diagnosis of malaria involves microscopy with visualization of Giemsa-stained parasites in a blood sample.[10],[11] This requires access to a lab with the technical expertise to identify malarial parasites and sophisticated microscopes.[12]We aim to create a portable diagnostics kit that will capture an image of a blood smear using a smartphone and test for the presence of malaria parasites using a Web API, making the diagnosis of the disease simple and efficient.
We have created a deep learning software based on Convolutional Neural Networks (CNN). This software will be able to identify the presence of malarial parasites from images of blood smears. The model has been trained to identify infected samples using a data set of twenty-seven thousand images of blood smears that have been identified by experts to be positive or negative for the presence of Plasmodium falciparum parasites. To capture the images of the blood smears, we plan to use a foldscope (an origami-based paper microscope) and a cost-effective centrifuge, developed by Dr. Manu Prakash.
Sponsors and References
References:
- Hanssen, E., Goldie, K. N., & Tilley, L. (2010). Ultrastructure of the Asexual Blood Stages of Plasmodium falciparum. Methods in Cell Biology, 93–116.
- Team:Heidelberg - 2014.igem.org. (2014).
- Lennartz, F., Adams, Y., Bengtsson, A., Olsen, R. W., Turner, L., Ndam, N. T., … Jensen, A. T. R. (2017c). Structure-Guided Identification of a Family of Dual Receptor-Binding PfEMP1 that Is Associated with Cerebral Malaria. Cell Host & Microbe, 21(3), 403–414.
- Lennartz, F., Smith, C., Craig, A. G., & Higgins, M. K. (2019). Structural insights into diverse modes of ICAM-1 binding by Plasmodium falciparum-infected erythrocytes. Proceedings of the National Academy of Sciences, 116(40), 20124–20134.
- Hsieh et al. (2016). The structural basis for CD36 binding by the malaria parasite. Nature Communications.
- Wright, K. E., Hjerrild, K. A., Bartlett, J., Douglas, A. D., Jin, J., Brown, R. E., … Higgins, M. K. (2014). Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies. Nature, 515(7527), 427–430.
- Lau, C. K. Y., Turner, L., Jespersen, J. S., Lowe, E. D., Petersen, B., Wang, C. W., … Higgins, M. K. (2015). Structural Conservation Despite Huge Sequence Diversity Allows EPCR Binding by the PfEMP1 Family Implicated in Severe Childhood Malaria. Cell Host & Microbe, 17(1), 118–129.
- Cybulski, J. S., Clements, J., & Prakash, M. (2014). Foldscope: Origami-Based Paper Microscope. PLoS ONE, 9(6), e98781.
- Bhamla, M., Benson, B., Chai, C. et al. Hand-powered ultralow-cost paper centrifuge. Nat Biomed Eng 1, 0009 (2017)
- Malaria Diagnosis Using a Mobile Phone Polarized Microscope. Casey W. Pirnstill, Gerard L. Coté. Sci Rep 5, 13368 (2015).
- Imaging and identification of malaria parasites using cellphone microscope with a ball lens. Temitope E. Agbana, Jan-Carel Diehl, Fiona van Pul, Shahid M. Khan, Vsevolod Patlan, Michel Verhaegen, and Gleb Vdovin
- Center for Disease Control and Prevention; Malaria diagnostic tests (February 19, 2020);
Results
Modeling
Inhibitory peptide sequences against Plasmodium falciparum were finalised after the analysis of multiple molecular dynamics(MD) simulations. Below are two of the shortlisted Inhibitor sequences grafted on Kalata B1 cyclotide. These are sequences that show lowest interaction energy and are based on the interactions of the parasite within the bloodstream stage of its life cycle.
Diagnostics
DeLeMa Detect is a cost-effective, easy to use and portable diagnostic solution. Our diagnostic kit analyses the blood smear images with the help of deep learning algorithms from a database of 27,000 images with an accuracy of 95.45%.Wet Lab
We cloned the Human Protein of the PDB-5LGD interaction Platelet Glycoprotein 4 using Restriction free cloning.[Image 1] depicts the confirmation of the Megaprimer (Insert sequence along with the extremities complementary to the plasmid vector of interest ~ 1500kb).
[Image 2] depicts the final cloned product of Restriction free PCR using the Megaprimer described above. The Plasmid vector used was pET28-a(+).
