Team:IISER Kolkata/Human Practices

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Integrated Human Practices

Guidance from Dr. Rupak Datta

The first step of our project was to build a solid theoretical foundation, based on practical considerations. Dr. Rupak Datta of the Department of Biological Sciences at IISER Kolkata specifically uses Leishmania as a pathogenic model in his research. We turned to him to gain an expert opinion on whether our model was rational and achievable.

• Targeting Iron is the correct decision

In order to effectively eliminate the parasite, we had to first locate aspects of its physiology that could be targeted. One of our primary options was the requirement of iron by the parasites for survival.

Dr. Datta, having worked extensively with several parasites including Leishmania, helped us understand the physiology of Leishmania and locate its weaknesses. He confirmed that iron was one of the biggest factors on which Leishmania was heavily dependent, and would be the most logical pathway to tackle.

• ROS burst is unpredictable, and macrophage has regulatory mechanisms

One of the killing mechanisms we were considering, was the release of a Reactive Oxygen Species (ROS) burst by UnLeish within the macrophage. However, after discussion with Dr. Datta, he explained that not only is an ROS burst difficult to control, but the macrophage also has extremely efficient defences against oxidative damage. This led us to further research, where we found that several mechanisms such as the NRF2 (nuclear factor erythroid 2-related factor 2) pathway would come into play in the case of ROS burst, as well as the fact that it could cause unforeseen damage within as well as outside the cell. Eventually, this led us to discard this alternative and focus the model on iron deprivation.

• Bacteria being inserted into the bloodstream should be completely safe

We broached the topic of studies on bacterial therapy, where bacteria are being used for efficient targeting of disease-specific regions in cancer, and how the same idea can be implemented in the case of anoxic Leishmania-infected regions in the spleen. Despite being initially wary, Dr. Datta agreed that our solution was viable, but emphasized the need to ensure that it would be a safe treatment. The best choice of the bacterium for the projected therapeutic application of our system is one that infects cells efficiently and can survive well inside the macrophage. However, such a bacterium will also be a potential hazard.

This advice led us to be mindful of using a bacterium that was or had been rendered non-pathogenic, while still being able to persist inside the cell long enough to act against the Leishmania. To test our genetic circuits and for experimental ease, we used E. coli DH5-α. However, for UnLeish, we would need a bacterium that could survive and replicate well within the macrophage. We eventually found the perfect candidate - E. coli O157:H7. Since this bacterium inherently produces Shiga toxin, the gene for Shiga toxin would be knocked out.

• Choosing the right parasite

Despite Leishmania donovani being the parasite that our model aims to target, Dr. Datta suggested we use Leishmania major for our experiments. This was primarily due to the fact that L. donovani is highly infectious and requires laboratory experience and the requisite safety equipment.

Meet the oracle

Every project needs a sanity check to analyze its feasibility, and to make it conceptually sound. We sat down with Mr. Sourav Banerjee, a Senior Research Fellow working in the lab of Dr. Rupak Datta to seek his guidance. His work revolves around the protozoan parasite Leishmania.

Here is an account of what we learnt and how it was integrated into the project:

• Consolidating Iron as the target for UnLeish

Not only is iron essential in DNA repair, synthesis of enzymes and for the metabolic process of the Electron Transport Chain (ETC), iron also plays a critical role in the transformation of Leishmania from the non-infective promastigote stage to infective amastigote stage. This makes the Leishmania parasite heavily dependent on iron. This was in agreement with our hypothesis that restricting iron availability for Leishmania will affect growth and replication of the parasite.

• Put to the test : Iron deprivation of Leishmania

To test the above hypothesis, we designed and conducted experiments with Leishmania parasite. For safety purposes, our experiments were conducted on Leishmania major, and not Leishmania donovani. We found out that Leishmania is indeed into the ‘heavy metal’ genre! The details of the experiments are given in the results section.

• Real World meets Mathematical modelling: Leishmania can modulate the iron homeostasis of the macrophage to suit its purpose

We gained the vital information that the parasite can stop the iron export from macrophages to the extracytoplasmic space. This led to the crucial realisation in the mathematical modelling that after being infected by Leishmania for a certain time, the iron concentration does not decrease due to macrophage iron export processes.

Ferroportin is the only iron exporter known in vertebrates and it functions to export iron from the cellular cytoplasm to outside the cell. We learnt that a study by Dr. Norma Andrews, at the University of Maryland, showed that when macrophages are infected by Leishmania , it eventually causes the degradation of ferroportin. Suspension of ferroportin activity leads to the internalisation of the iron. There occurs an increase in the cytosolic labile iron pool which can be acquired by Leishmania. This is an ingenious method by which the parasite tries to acquire more iron.

• There are several ways in which Leishmania can acquire iron in the phagolysosome

Leishmania resides inside the phagolysosomal compartment of macrophage cells and cause infection. Macrophages are major players in the recycling of senescent RBCs. As a consequence of phagocytosis of senescent RBCs the haem component in them is released. Leishmania cannot synthesize haem by themselves but by this process they can get haem from the phagolysosomal environment. Other sources like haptoglobin or even free haem in the bloodstream can also act as a source of iron for the parasite

• Supporting information to the fact that Leishmania can lower Nitric Oxide (NO) concentration inside the macrophage, which helps the parasite survive

NO is an effector of the immune system and helps us inhibit infections. Infection by Leishmania major stimulates the production of inducible nitric oxide synthase (iNOS). These enzymes eventually produce nitric oxide. This NO has a detrimental effect on the survival of the parasite. In L. amazonensis, HDAC1 suppresses the production of iNOS so they cannot generate the nitric oxide, which enables the parasite to survive within the macrophage.

A Talk with Dr. Nahid Ali

Once we had created the basic framework of our project, it was time to understand the ground realities of the disease that we had taken on. We visited CSIR-IICB (Council of Scientific and Industrial Research – Indian Institute of Chemical Biology) in Kolkata, and met Dr. Nahid Ali, a leading scientist of the country working in the field of Leishmaniasis.

• Kala Azar occurrence shows a cyclical nature, and an epidemic might reoccur

After Dr. Ali had understood the details of our project, she briefed us about the status of the disease across the globe. She told us that even though incidences of Leishmaniasis have decreased due to active government plans to combat the disease, the decrease can also be attributed to the cyclical nature of the disease. Previous statistics have shown that the disease prevalence shows a decadal increase-decrease cycle, so it cannot be said with certainty if the decrease is due to effective treatment or due to this cycle.

• A hidden reservoir of parasites that could lead to an outbreak

Another problem that has surfaced is the number of increasing Post Kala-Azar Dermal Leishmaniasis (PKDL) cases, a glaring problem which requires immediate attention. PKDL patients are individuals who have been successfully treated for Visceral Leishmaniasis, but the parasite resurfaces in the form of skin lesions. These do not respond to conventional treatment and are a potent reservoir of the parasite. For example, in African nations, almost 50% of patients previously infected with Leishmania develop PKDL! All these factors together can come together and lead to an outbreak of epidemic proportions. According to her, unless the incidence of the disease reduces to below 1 in 10,000 individuals, the disease should be considered an extant risk, and measures should be continually taken.

• Current drugs are dangerous, distressing and expensive

We then discussed with her the effectiveness of the treatments currently available in the market. She told us about Pentavalent Antimonials, which were the drugs of choice for decades in the Indian subcontinent. But not only did these show toxic side effects, the high usage led to a widespread resistance in the parasite against these solutions.

Amphotericin B, another compound used against Leishmaniasis is also toxic and requires close medical attention for a long period of time. In her words, “Amphotericin B somehow stimulates macrophages, activating the immune system, and makes it less susceptible to future infections (like PKDL). Another form of the same drug, Liposomal Amphotericin-B (AmBisome®) , possesses higher efficacy and lower toxicity, but does not lead to the above ‘stimulation’ of the macrophage, leading to weaker activation of the immune system and thus reduced resistance of the macrophage against future infections.” The mechanism for this is not well understood. Increasing use of AmBisome® has led to an increase in cases of PKDL, which is a cause of concern, according to Dr. Ali. The drug Amphotericin-B and its liposomal counterpart are also very expensive. In India and a few other countries, the medicine has been subsidised due to government aid. However, patients in other nations like those in Africa, may not have ready access to this medicine due to its high cost. And now, there are increasing reports of resistance to this drug as well.

• At present, we should not direct our project towards diagnosis, as more efficient methods exist

We then inquired about the present diagnostic methods for Leishmaniasis, and their performance. According to her, the most accurate test for the disease is the highly invasive ‘splenic aspirate’ method, whereby a biopsy of the infected organ is taken and tested. Many other less invasive methods are being developed. A certain laboratory has been working on a dipstick test, which can identify certain pathogen-specific antibodies in the blood. She herself is working on a test device which can sense similar antibodies in the urine, making diagnosis totally non-invasive. But it will take time before these products are available to the general public, and they will carry a substantial price tag.

• Nitric oxide concentration change is a cell-wide event

After these discussions, we got some conceptual and experimental doubts regarding our project clarified. We found out that the change in nitric oxide concentration after infection with leishmaniasis is a global event, i.e. it is a change that is not limited the Parasitophorous Vacuoles (PV) but is present everywhere inside the macrophage. This information was crucial for the viability of our project since UnLeish will first sense the NO level within the cell, and then attack.

• Our UnLeish-containing endosomes will almost certainly fuse with Leishmania-containing PV

She also confirmed that our bacteria will probably be able to reach the PV where the Leishmania reside, by fusion of the bacteria-containing endosome with the PV. This is essential, as it will provide a vantage point for UnLeish to produce its effect and eliminate the parasite effectively.

All the knowledge we gained from our interaction with Dr. Nahid Ali, led us to the conclusion that leishmaniasis is not a disease of the past, and cannot be ignored. The government and the medical community must be vigilant and work together to prevent any future outbreaks of the disease so that we can look forward to a Kala Azar-free future.

Village interaction

Integrated Human Practices is about imbibing information from not only the expert, but also the affected. To understand the nature of the treatment that we needed to develop, it was crucial to interact with the population that the treatment was meant for. We achieved this through an extensive and detailed interaction with the residents of Dalaipur village in an area of West Bengal which, as explained by Dr. Nahid Ali, had a history of leishmaniasis cases.

Our experience in the village allowed us to understand the population at risk and their requirements, as well as witness their living conditions. We realised that we had to focus on creating a treatment with minimal side effects and minimal time spent under medical care, at the least possible cost. This is how we decided to ensure that only the infected macrophages of the body would be targeted, minimising damage to the individual’s immune system.

Our discussion with the village inhabitants also gave us vital information as to the knowledge that was lacking, and the aspects of the disease that we needed to further inform them about - this led to the creation of our nukkad naatak, or Street Play.
For a more detailed account, please take a look at Village Interaction.