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A novel insight into the mechanism behind what makes Tuberculosis tick

An increasing number of cases of multi-drug resistant tuberculosis and total-drug resistant tuberculosis are being discovered in India, accounting for the highest TB burden in any country across the globe. It has been said, ‘It is not the strongest species that survive, nor the most intelligent, but the ones most responsive to change’. Mycobacterium tuberculosis is able to adapt and thrive despite the immune system and drugs that are targeted against it. This is due to its ability to sense and adapt to its host's environment. Dr. Deepak Saini’s laboratory at the Indian Institute of Science, Bangalore, is working at unraveling the molecular mechanism that helps M. tuberculosis sense, respond and adapt to its host environment.

Starting with the basics, for any sensory system to function, there has to be a minimum of two components with one receiving the signals and then subsequently transferring the signal to another component.  Two-component signaling, as the name suggests has two protein components involved in the signaling.  The first is a ‘receptor’ molecule called the Sensor Kinase (SK), which is present on the outer membrane surrounding cells and is responsible for ‘receiving’ diverse environmental stimuli. The second component is the ‘effector’ molecule called the Response Regulator (RR), which when activated by the Sensor Kinase (SK) produces an ‘effect’, by binding to specific regions of the bacterial DNA and activating the decoding of genes involved, in the response to that stimulus.

“You can, therefore, appreciate the extreme importance of these systems for the pathogen”, explains Dr Saini. “Without these systems, the pathogen will not be able to adapt and survive inside the hostile and constantly changing host environment. Understanding signaling systems like the two component system may be the key to combating this pathogen more effectively”, he adds.

Interactions such as those that occur between RR and SK are extremely short-lived and thus very challenging to study. The most common way to study transient interactions between two proteins is to use proteins with a radioactive tag which are neither easy to use nor is safe. In a recent study published in Biochimica et Biophysica Acta-General Subjects,researchers from Dr. Saini’s lab have described a non-radioactive method to study SK-RR interactions using FRET.

Fluorescence Resonance Energy Transfer (FRET) is a mechanism in which energy is transferred between one light-sensitive molecule to another. In this instance, the two proteins that we are interested in- SK and RR are linked to two different molecules (let’s call them, F1 and F2) that can fluoresce, but at different energies. When we hit SK-F1 and RR-F2 molecules with light of certain energy, F1 (let’s suppose) lights up. The energy released upon F1 lighting up, can light up F2, provided (and, this is the imported part) SK and RR are at an extremely close (interacting) distance. So, every time F2 lights up, we know that SK and RR must have interacted.

Using this technique, the group demonstrated that SK and RR molecules interact in the non-activated state. When SK is activated, it adds a phosphate group to RR which causes a decrease in the affinity between the two. The study has helped provide evidence to prove this phenomenon which was only a hypothesis until now, and thus improve our understanding of how these proteins work.

Previously, it was thought that one type of SKs adds a phosphate group to only a single type of RRs. In this study, however, it was discovered that some SKs add phosphate groups to more than one type of RRs. The rate at which some SKs add phosphate groups to their corresponding RRs was also discovered to vary between different SKs.

The current technique, however, studies SK-RR interactions outside of the cell and not in its natural environment. Dr. Saini hopes to adapt this technique to study these proteins inside the cell, in real time. This will provide further insight into the complexity of the sensory system, its function in a biological system and tell us more about the molecular mechanisms behind it.

“If you think of this in terms of host-pathogen interactions, and the ability of M. tuberculosis to survive inside the host, the fact that we have found a possible link between the intrinsic biochemistry of the two component system protein interactions and their ability to form a vast cross-communication network encourages us to push further into studying this interesting signaling system.The future prospects are very exciting,” signs off Dr. Saini.

About the authors

1] Ruchi Agrawal is a Ph.D. student in the Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science

2]Prem Kumar V. is with the Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science

3] Harini Ramanan is with the Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science

4] Deepak Kumar Saini is an Assistant Professor at the Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science


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About the paper

Title:  FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis

Publication: Biochimica et Biophysica Acta (BBA) - General Subjects