Team:IISER Kolkata/Design

Design

Overview/Introduction

Our aim is to create a therapeutic against infections caused by the Leishmania parasites. The therapeutic agent must be able to -

  1. Target only the infected cells,
  2. Successfully treat the infection with minimal side effects,
  3. Not be susceptible to resistance,
  4. Be able to be contained after successful treatment.

This led to the creation of a genetically modified bacterium - unLEISH, which could sense NO (Nitric Oxide) levels inside a macrophage, to detect infection and administer a therapeutic agent (Aerobactin - an iron chelator), to deprive the parasite of Fe (iron), possibly followed by its death.

Leishmania and the art of evasion

Leishmania is a specialised parasite. Millions of years of evolution has helped it establish its own niche inside the animal macrophages. A Leishmania parasite employs a variety of techniques to escape immune responses. One such technique is downregulation of NO. Leishmania parasites are reported to downregulate Nitric Oxide levels inside a macrophage by inhibiting the iNOS production.[1]

unLEISH exploits this technique used by Leishmania against it.

unLEISH 

“The world is full of obvious things which nobody by any chance ever observes.”

― Arthur Conan Doyle

NO (Nitric oxide) is a good candidate for a disease biomarker. E. coli has well characterised promoters which responds in presence of different concentrations of NO. We knew that Leishmania can downregulate the NO levels, but it was also evident that the NO levels still stay higher than a basal NO levels of an uninfected macrophage[1]. Moreover, after the treatment was over and the parasite was eliminated, NO levels would rise again in the absence of down-regulation, and the treatment would no longer be required. Thus, we wanted a dynamic switch that would work in a ‘range’ of NO concentrations, and would be switched on only when the NO levels are intermediate.

unLEISH possesses a genetic circuit with two NO promoters having different binding affinities to NO. The promoter with high binding affinity senses lower NO levels, thus it switches the genetic circuit on after a certain NO concentration, which is achieved after Leishmania infection. The promoter with low binding affinity senses very high NO levels, thus it switches the genetic circuit off after a high NO concentration is reached.

NO Sensing : The workings behind the superpower

So, how did we construct a sensing circuit that would activate in a range of Nitric Oxide concentration? The key is to have two different promoters with different affinities to NO, as we explained above.

This is how the circuit looks like -

The points below explain the workings of the components.

  • NO detector 1 is an inhibitor, has a high affinity for NO, and ceases to inhibit when NO is bound to it.
  • NO detector 2 is an activator, has a low affinity for NO, and it activates only when NO is bound to it.
  • TetR protein is Tetracycline Repressor Protein.

Also, the binding sites have been colour coded for convenience.

CASE 1:

Suppose the NO concentration is pretty low, that is when the unLEISH is being grown in medium or in tissue spaces which do not have any NO.

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As it is seen in the images, NO detector 1, which produces a transcriptional inhibitor, binds to its operator site in the near absence of NO. The NO detector 2, is a transcriptional activator, which works only if NO is bound to it, is also inactive.

CASE 2:

Suppose the NO presentation is too high. This situation arises when unLEISH enters a macrophage which is not infected, which causes the NO levels to shoot high.

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In such a case, the circuit remains inactivated.

CASE 3:

Now, let’s take a look at what happens when the NO concentration is intermediate, i.e., when Leishmania has infected a macrophage and downregulated the NO level.

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Here, due to the fact that NO detector 1 has a higher affinity, it binds to the NO present and stops repressing the circuit. NO detector 2, having a lower affinity, cannot bind to NO and cannot activate TetR, which will go on to repress the circuit.

So, what we have is a circuit, which can sense NO levels and can activate itself in a certain range of NO concentrations. All we need are NO detectors which do the job, and providentially, we found two NO promoters which fit our needs.

NO detector 1 - NsrR is a candidate for this. It’s an ‘HTH (helix-turn-helix) type transcriptional repressor’.[2]

NO detector 2 - NorR, a transcriptional activator for the norVW operon.[3]

Now that we’re done with sensing, let’s take a look at what makes our unLEISH an awesome assassin!

Leishmania and Iron : The bond of ages

Leishmania and Iron have a relationship that has existed for time immemorial.One of the fundamental steps in the development of a pathogen within the host is acquiring Iron, although, host cells are known to implement several iron sequestration policies to encounter the different invasion strategies of pathogens. In our case, Leishmania requires the iron acquisition for growthand to combat oxidative stress because of the presence of iron-containing antioxidant enzyme superoxide dismutase (Fe-SOD). Inactivation of Fe-SOD negatively affects intracellular survival and virulence of the Leishmania [5]

Our unLEISH, being a smart killer, targets this very aspect of Leishmania’s existence, Iron.

Let’s see how.

unLEISH and its ironical fate

Leishmania is a clever pathogen, it has evolved various ways to intake iron in the hostile environment of the macrophage’s Parasitovorous Vacuole (PV). But our unLEISH is smarter, as it reduces the amount of iron directly at the source, i.e, the Labile Iron Pool of the macrophage. It achieves this feat by using an iron chelator, a molecule which binds to Iron to form a complex with it, and render it useless for the Leishmania.

unLEISH is equipped with a very strong iron chelator, Aerobactin, which, as it is shown before, is under the control of the dynamic NO sensor. Originally used by certain strains of E. coli to sequester iron from iron-poor environments, unLEISH uses it to chelate iron present in the Leishmania microenvironment, which deprives the parasite of iron, and eventually, eliminates the parasite.

Irresistible, ain’t I?

The following facts ensure that Leishmania does not develop resistance against unLEISH.

  1. Iron is a very important element for the parasite, and there’s no alternative to it, thus, Leishmania cannot evolve to survive without Iron.
  2. Aerobactin is a strong Iron chelator, it’s very difficult for Leishmania to develop a system to liberate iron from Aerobactin.

The aftermath

After the successful elimination of the Leishmania parasite from the macrophage, the NO levels will rise as there’s no downregulation anymore, and due to the presence of unLEISH, which is a bacterium. The risen NO levels will now activate the immunological machinery of Leishmania, which will lead to its action against unLEISH, leading to the death of our beloved unLEISH This also brings us to the question of the chassis, to be chosen for the project.

Chassis!

The idea of using bacteria as therapeutics is not entirely new. Bacteria have prospected as vessels for targeted cancer therapy[6]. For the proof of our model and checking all genetic circuits outside the macrophages, we are using E. coli DH5α as a chassis. But it cannot survive for long inside a macrophage. Choosing the ideal chassis suitable for our project is a daunting task, as the chassis should

  • Survive and reproduce inside the hostile environment of a macrophage phagolysosome.
  • Not cause any adverse effect to the patient being treated.

A possible candidate for the job is E. coli O157:H7, which produces Shiga Toxin. The gene which produces this toxin can be knocked out.

References

  1. Srivastav, Supriya & Ukil, Anindita & Das, Pijush. (2015). Elucidating the Strategies of Immune Evasion by Leishmania. 10.21775/9781908230522.06.
  2. UniProtKB - P0AF63 (nsrR E. coli)
  3. UniProtKB - P37013 (norR E. coli)
  4. Meyrier A (1999). "Urinary Tract Infection". In Schrier RW, Cohen AH, Glassock RJ, Grünfeld JP. ISBN 0-632-04387-3.
  5. Adak, Subrata, and Rupak Datta. Leishmania Current Biology and Control. Caister Academic Press, 2015.
  6. Van Dessel N1, Swofford CA, Forbes NS.(2015) Potent and tumour-specific: arming bacteria with therapeutic proteins. DOI:10.4155/tde.14.113