Description and Inspiration

“Unavailability of a vaccine and limited chemotherapeutic options coupled with the rapid emergence of drug-resistant Leishmania strains challenges researchers to come up with novel strategies to combat the disease”

-Subrata Adak and Rupak Datta [10]

What is Leishmaniasis?

Figure 1 - How different forms of *Leishmania* causes damage

Leishmaniasis is caused by a protozoan parasite from over 20 Leishmania species. Leishmania is able to persist in host cells by evading or exploiting host immune mechanisms. The parasite lies exclusively intracellularly, mainly inside macrophages as replicating amastigotes. These amastigotes multiply and burst out of these macrophages and infect other cells. There are 3 main forms of leishmaniasis – visceral (also known as kala-azar and the most serious form of the disease), cutaneous (the most common), and mucocutaneous. The following flow chart explains the process through which the parasite wreaks havoc.

Figure 2 - Lifecycle of the parasite in the human body

The Problem

It is the world's second most deadly parasitic disease after malaria, in terms of mortality [1]. The World Health Organization data indicates that an estimated 700,000 to 1 million new cases and 20,000 to 30,000 deaths by leishmaniasis occur annually in the world.

Categorized as a Neglected Tropical Disease - a diverse group of communicable diseases that prevail in tropical and subtropical conditions in more than 149 countries, and are relatively overlooked by developed nations. These affect more than one billion people and cost developing economies billions of dollars every year.

The disease devastates some of the poorest people on earth and is associated with malnutrition, population displacement, poor housing, a weak immune system and lack of financial resources. It is also linked to environmental changes such as deforestation, the building of dams, irrigation schemes, and urbanization etc [2].

Different forms of leishmaniasis and its spread across the world:

Table 1
Type	Countries affected	Severity
Visceral leishmaniasis	Brazil, Ethiopia, India, Kenya, Somalia, South Sudan and Sudan	50,000 to 90,000 new cases
Cutaneous leishmaniasis	Afghanistan, Algeria, Brazil, Colombia, Iran and the Syrian Arab Republic	600,000 to 1 million new cases
Mucocutaneous leishmaniasis	Bolivia, Brazil, Ethiopia and Peru	90% of mucocutaneous leishmaniasis cases

India, Nepal, and Bangladesh have more than 50% of the global burden of visceral leishmaniasis. In 2005, these countries committed to eliminate visceral leishmaniasis as a public health problem, but another outbreak occurred in 2005. The projected deadline for this eradication was 2015, but this deadline is far from being met.

A Local Perspective: Visceral Leishmaniasis (VL) in India

Ever since its first documentation by military surgeon William Twining in the 19th century, visceral leishmaniasis (locally known as kala-azar) has been a problem in India [3]. At present, it is a serious public health problem in Indian subcontinent. In India, kala-azar is endemic in Bihar, Jharkhand, West Bengal (Our state) , and Uttar Pradesh, making it an issue which is also of greater relevance to the community here at IISER Kolkata because we are at the epicentre of this problem.

Why tackle a solved problem?

From the outlook, the eradication programs look like a success on paper, but from our extensive literature survey, iGEM IISER-Kolkata team found out that there are some concerning information that could be detrimental to the eradication program.

Epidemiological pattern:

Even though the programme appeared to have contributed to a global decline of VL, but significantly, the disease commonly shows a cyclical epidemiological pattern, as depicted in the figure below. The parasite is known to be drug-resistant (antimonial drugs are not effective in many areas of India due to over-use) and hence we may in a position where a resurgence of kala-azar can occur [3]

Figure 5 - Kala-azar cases in India during the period from 1987 to 2011 (source: Directorate General of Health Services, National Vector Borne Disease Control Programme (NVBDCP), Government of India, and State Health Society, Bihar, India). ( Source [12] )

Deadly Treatment:

There is no vaccine available for VL; hence control of VL exclusively depends on chemotherapy. Liposomal Amphotericin B, Paromomycin and Miltefosine are currently widely used for this purpose. But these treatments have toxic side-effects (shown in the table below) that ironically required other treatments and sometimes hospitalisation [4].

Table 2
Medicines	Side Effects
Liposomal Amphotericin B	Infusion related fever and rigor Chills Vomiting Backache
Paromomycin	Ototoxicity Nephrotoxicity
Miltefosine	Gastrointestinal effects (vomiting, diarrhea, abdominal pain) (major) Nephrotoxicity (decreased urine, renal failure) (major) Hepatotoxicity (jaundice) (major) Any other, unanticipated (edema, anemia)
Amphotericin B	Fever with chills and rigors (major) Vomiting, dehydration (major) Edema, decreased urine, renal failure Arrhythmias (major)

And costly too!

The cost of treatment is important when patients need to pay for treatment as ~75 % of the VL cases in Bihar live below the poverty threshold of less than US $ 1.0 a day, and this is similar in other endemic countries although exact data are scarce [5]. Poverty seriously affects the prognosis of VL because most of the patients and their families have to pay for diagnosis, drugs and hospital care, and this is often half or more of the annual household income [6]. As a result, families with a VL infected member descend deeper into poverty [7].

Post Kala-azar Dermal Leishmaniasis (PKDL):

Post kala-azar dermal leishmaniasis is a complication of VL. it is characterised by a macular, maculopapular, and nodular rash in a patient who has recovered from VL and who is otherwise well [8]. PKDL probably has an important role in interepidemic periods of VL, acting as a reservoir for parasites. The alarming fact is that PKDL can occur in VL cured patients within a period of few months to many years!

The eradication program fails to keep the number of cases of PKDL down. While then number of VL cases are decreasing every year, the number of PKDL cases is increasing. From only 499 cases in 2013, it jumped to 1982 cases in 2017 and there seems to be no stopping [9].

Our Solution: UnLeish

We plan to do this by genetically engineering a bacterium which can sense a Leishmania major-infected macrophage using a Nitric Oxide (NO) sensor. In response, it will express an iron chelator (Aerobactin) to efficiently kill the Leishmania parasites.

Why Nitric Oxide?

Nitric Oxide (NO) is an intracellular secondary messenger which regulates various physiological functions. The generation of reactive oxygen and reactive nitrogen species, in response to pathogen attacks, play a central role in host (macrophage) defence mechanism. The Leishmania parasite cleverly evades the assault by evolving several defence strategies. We exploited one of these mechanisms to detect a Leishmania-infected macrophage [10].

Whenever a pathogen is phagocytosed into a macrophage, nitric oxide levels inside that macrophage rise drastically up (C2) as an immunological response from basal levels (C1). But, the parasite has the ability to down-regulates this increase and brings it to a lower level because NO is not only a reactive oxygen species, but also it plays a major role in activating other immunological response that is detrimental for survival of the parasite.

Our bio-sensor detects this downregulation of NO and in the process senses the parasite-infected macrophage.

Elimination by Chelation:

Iron is a vital nutritional requirement for virtually all organisms, including pathogenic trypanosomatid parasites (Leishmania), and plays a crucial role in many facets of cellular metabolism as a cofactor of several enzymes. Iron acquisition is essential for the survival of parasites. In the macrophage, Leishmania tampers with the iron regulation system and increases the intake of iron so that it can utilise a part of it for its survival [11].

After our biosensor detects a parasite-infected macrophage, our proposed GMO plans to chelate all Iron inside the macrophage by producing an Iron chelator. This will severely hamper the growth and reproduction of the parasite and subsequently eliminate it.

Figure 7 - Diagram illustrating our GMO in action. It only produces Aerobactin when it senses the parasite infected macrophage