This interview is a part of the series THSTI SciSpeak where the spotlight is on the work being done at THSTI. Through this feature, we intend to reach out to students aspiring to pursue a career in science in India by getting our scientists to answer questions on their choice of a scientific career, the field they chose, what worked and did not work for them and more.
Curiosity will kill the cat but made a scientist of a boy.
Dr. Nisheeth Agarwal began what is one of the first TB labs at THSTI and currently heads the mycobacterial genetics laboratory. He joined the institute in 2010 and has had 6 research fellows, 6 PhD students and many short-term trainees since then. He wears a rough exterior and so I was in for a surprise at the answer to a ‘warming-up’ question of ‘what motivated him to be a scientist”. He said, “Curiosity begets an investigative mind. Right since I was a kid, anything less known triggered curiosity. The first thing I would do with a new toy was unscrew them to look inside and since I wasn’t skilled enough to assemble them back, they were all gone too soon (chuckles). The downside is some of my folks still remember how I tricked some of their gadgets such as radio transistors, alarm clock etc. Nonetheless, the urge to explore was inherent and exploring and understanding things around me only got more passionate with age. This probably seeded the scientist in me (which I didn’t realize then).”
He recalls, “My dad inspired me to become a clinician, but a bad memory impeded from qualifying any national pre-medical examinations. I felt I should instead channelize all the ‘passionate curiosity’ into a career in science. I joined the master’s degree course at the School of Biotechnology, Banaras Hindu University (BHU) while it was still an upcoming subject two decades ago. And no, it wasn’t like I already had a plan. It wasn’t till I met Dr. Anil Tyagi, a faculty member at Delhi University South Campus, during his visit to BHU that I decided to take up TB research. His laboratory focused on various aspects of TB, and since I have personally experienced few TB cases in my family, the choice of venturing into TB research only felt worthwhile.”
The TB program at THSTI works with the mandate of translating discoveries to provide effective and affordable solutions and has been designed accordingly by bringing together scientific expertise. Nisheeth ventured into TB research right when he was a doctoral candidate at DUSC and is one of those people whose career trajectory took him to different places (read laboratories) with Mtb always waiting for him like an unfinished business. He describes, “The major objective of my doctoral thesis was to understand how gene transcription is regulated in Mycobacterium tuberculosis (Mtb). Transcription of DNA into RNA is the first step of flow of information embedded in genes into proteins. An important actor is the multi-subunit enzyme RNA polymerase which recognizes specific DNA sequences known as promoters when aided by the sigma factor (a protein subunit). A year before I began my work, the Mtb genome was sequenced revealing the presence of 13 sigma factors. I was fascinated by how the principal sigma factor, SigA specifically recognizes the cognate promoter sequences (recognition site on the DNA sequence) in the genome. My work led to the identification of many genes that are transcribed with the help of SigA, essential for growth of Mtb and might have contributed largely to the success story of this dreaded microorganism. We thought that some of these could be explored as anti-TB drug targets (read about these findings here)”.
Nisheeth was resolute about the fact that his work had only begun, and he needed to delve deeper. “While working on the doctoral research project, I was determined about continuing investigations in this area as there was a lot more to explore and know about the pathogen and its pathogenesis.” Nisheeth went on to work at one of the best laboratories in the world working on molecular pathogenesis of Mtb for his postdoctoral training. “I joined Prof. William R. Bishai at the Johns Hopkins University in 2005 and studied multiple aspects of Mtb pathogenesis (read about their work here, here and here). I could, as a result of this collaboration, identify the role of an extracytoplasmic function (ECF) sigma factor SigM (read more about the ECF sigma factors here), and the WhiB1 transcription factor in Mtb physiology (you can read about these findings here and here). I also observed and was surprised that Mtb overproduces a secondary messenger molecule, known as cyclic AMP (cAMP) which is secreted inside host cells during infection. We could establish for the first time that the Mtb-derived cAMP origin intoxicates host and protects the pathogen from the host rebuttal by modulating the host signalling pathway (read this work here). This work was important as it highlighted that signal transduction interference adopted by Mtb is a possible new target for anti-TB drugs.”
After completing his postdoctoral stint at JHU, he decided to move back to India with the ambition of setting up his own research laboratory turning down a lucrative offer to stay back at the USA and independently lead the research group. The passion to serve one’s own country where TB was taking lives everyday outweighed that offer.
Back in India
While passion to serve one’s country is great, but the scientist also needs a formidable question to move ahead. Nisheeth goes on to describe some that he was prepared to take head-on. “Some of the burning queries prevailing when I returned were, ‘why Mtb grows so slowly (duplication time of ~21hrs as against Escherichia coli with 20-30 mins); how a subpopulation of Mtb transforms into the daunting drug-tolerant persisters; can we develop a feasible approach for rapidly identifying genes that are essential for growth under regular culture conditions and transformation into the drug-induced persisters; can we characterize the function of hitherto unknown essential genes and explore their potential as anti-TB drug targets, particularly targeting the persisters that require 4-6 months of additional treatment in a clinical setup; how does host metabolism influence the disease and treatment outcome?”, he said enumerating these questions.
A decade (well almost!) of the mycobacterial genetics’ laboratory – Nisheeth Agarwal lab
Nisheeth’s lab at THSTI is the first one he set up after coming back to India and it has been nine years since his team is working to find answers to these questions. He adds, “most of our efforts have been directed towards improving how we understand the mechanism of formation of persisters and the crosstalk between Mtb and host macrophages. We believe our research questions are important as an in-depth understanding for them is required to combat the dreaded pathogen and not much is known yet. We badly needed a tool to rapidly manipulate Mtb genome. We were the first team in the world to implement the CRISPR-interference approach in 2015, immediately after its inception, for efficiently silencing gene expression in mycobacteria (read about the team’s work here). The tool has since been disseminated for application to more than 40 laboratories all over the globe and has resulted in multiple collaborations. We have assisted multiple investigators in creating knockdown strains targeting genes of interest in their studies (read two of the studies that resulted through these collaborations here and here).” Mentioning few important collaborators, he says, “In the last one year we could initiate two collaborative projects, one with CSIR-Institute of Microbial Technology (under review) and another with National Institute of Immunology (funded by DBT). These projects are aimed at characterization and identification of new drug targets including some of the membrane proteases. We are also working to characterize the host phosphoproteome profile using THP-1 derived macrophages; understand the role of chaperones associated with Clp proteolytic machinery and address the underlying mechanism of formation of Mtb persisters (read the paper here). Our projects put together will help identify new drug targets, mechanism to shorten the duration of treatment by addressing persisters formation and the role of host-derived pathways in regulation of intracellular growth of Mtb. We are going to hit the pathogen’s evasion strategy at multiple points.”
Its been 20 years since the Mtb genome was sequenced but for what is an old disease caused by an older microorganism, the enigmatic pathogen is still winning (see these statistics by WHO here). Considering this, Nisheeth elaborated few areas young researchers should investigate, “It remains to be understood how certain individuals are innately resistant against Mtb infection or development of active TB disease despite having experienced equal exposure.” He points out, “There would certainly be some host-derived pathways differentially active in these subjects that need to be identified and studied.”
When asked about what he would want to tell his own and all other students who aspire to lead a research team someday, he said, “Some of the pre-requisites before one decides to start a lab of his own are, 1) to frame the right question through extensive reading; 2) developing an independent thought process to frame novel research questions; 3) setting up collaborations and taking external opinion on research projects; 4) being open to quickly grasp new information introduced in the field and implement in one’s own research projects; 5) working with an attitude to reach to the level of near-perfection; 6) setting up milestones and taking the self-imposed pressure to attain these goals in a defined time-period; 7) working with a schedule maintaining full discipline and sincerity in meeting the timelines; 8) decent communication skills to better present one’s work and findings. These qualities would help if one takes on the challenging role of a Principal Investigator.” He went on to admit that getting a PhD did change him. He said, “It not only improved my scientific skills but also left a long-lasting effect on many other aspects of my life. I am more disciplined now. I realize how essential being organized in your work, scientific or non-scientific is. A PhD is held more accountable and, so, should be more careful about scientific accuracy while communicating his/her research to, again, scientists and non-scientists.”