University of Delhi, New Delhi
International Centre for Genetic Engineering and Biotechnology, New Delhi
Indian Institute of Science, Bangalore
One-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). The number of deaths due to HIV-tuberculosis (TB) association and the emergence of multi/extensively/totally-drug resistant tuberculosis have reached alarming levels. Central to the success of Mtb as a pathogen is its ability to persist within humans for decades without causing clinical symptoms and to then reactivate to cause full blown active infection. Interventions that identify and eliminate latent infection might break the cycle of disease transmission and reverse the TB epidemic. Current approaches for detection, prevention and treatment of latent TB are inadequate, and rational development of new tools has been limited by a poor understanding of the fundamental biology of latent TB. I have exploited several multi-disciplinary approaches, new to the mycobacterial field, to study mechanisms associated with Mtb latency.
During my Ph.D. research at the University of Delhi (India), I obtained a strong interest in tuberculosis pathogenesis and gene regulation. In my dissertation with Prof. Anil K Tyagi I studied and characterized genes involved in regulating the virulence of Mtb. These studies led to the discovery of a virulence regulatory locus (VirS-mymA) in Mtb . I was instrumental in developing technology to create gene knock-outs in Mtb. During the later years of Ph.D, I gained expertise in analyzing various aspects of Mtb biology such as studying polyketide and lipid anabolism. Detailed biochemical studies on Mtb cell wall associated polyketides were done in collaboration with Dr. Ram A. Vishwakarma at the National Institute of Immunology (NII). I established various collaborations with scientists working in the area of TB within India to expand my understanding of Mtb pathogenesis. I consider these collaborations an important resource to my success as an independent scientist, particularly in India.
My Ph.D. and postdoctoral research have been complimentary experiences. In the laboratory of Dr. Adrie JC Steyn at the University of Alabama at Birmingham (UAB), USA, my interests shifted towards Mtb latency and questions surrounding gene function, signal transduction and dormancy. When I joined Dr. Steyn’s lab, he and I decided that it would be mutually beneficial if I used my previous experience with Mtb to set up original projects that were outside the traditional thinking of the TB field to address specific questions unique to mycobacteria. In this way, I tested my abilities to initiate an independent research plan (and received independent funding from the New York Trust [Heiser foundation]) and provided the laboratory with the tools and systems necessary to address questions pertaining to Mtb dormancy, and gene function. Below are my major achievements during post-doctoral research:
Dissection of virulence pathways through protein-protein interactions:
Deciphering the protein interaction networks utilized by Mtb would be instrumental to unravel some of the mysteries associated with Mtb pathogenesis and facilitate development of vaccines and diagnostics. I developed a novel tool based on protein fragment complementation and utilized a native host (M. smegmatis) to explore Mtb protein-protein interactions (Fig. 1). I exploited this experimental system to provide insights into translational research areas pertaining to the development of vaccines and diagnostics. The 6-kDa early secretory antigenic target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10) from Mtb are two dominant targets for T cells in the early phases of infection and were recently utilized to successfully develop the novel QuantiFERON -TB Gold In-Tube detection system (Cellestis). In an attempt to amplify the inherent immunogenicity of ESAT-6 and CFP-10, we screened a Mtb genomic DNA library to identify proteins which interact with these antigens using M-PFC. Several novel proteins identified from this screen can potentially be used to amplify the immune response to ESAT-6 and/or CFP-10 . This patented technology was subsequently distributed to several national and international investigators to detect protein-protein interactions and is anticipated to significantly advance TB research.
Revealing the identity of the first Mtb intracellular redox sensor:
In 1995, Des Collin’s group (AgRsearch, New Zealand), reported that a single point mutation in the 4.2 domain of the principle sigma factor (SigA) was responsible for the loss of virulence in a member of the Mtb complex. Subsequently, in 2002, Adrie Steyn showed that a small transcription factor, WhiB3, specifically interacts with wt SigA, but not with the single point mutant. These results suggest that the loss of virulence due to the single point mutation in SigA could be the result of abolished interaction with WhiB3 (a homolog of a sporulation factor in Streptomyces). Interestingly, it was shown that disruption of WhiB3 in Mtb (Mtb?whiB3) does not attenuate Mtb for survival in mice; however, mice infected with Mtb?whiB3 survived significantly longer. These results demonstrated that active replication of the pathogen is not exclusively responsible for clinical disease and the loss of WhiB3 renders Mtb defective in the production of immunomodulators that influence host pathology and survival.
However, some fundamental questions regarding the role of WhiB3 in the pathogenesis of Mtb remained unanswered: 1) What is the mechanism of WhiB3 mediated virulence regulation? 2) Is WhiB3 a transcription factor? and 3) What are the bio-active immunomodulators controlled by WhiB3?. In 2005, I initiated a project to study the role of WhiB3 in the pathogenesis of Mtb. Using multi-disciplinary approaches such as Electron Paramagnetic Resonance (EPR), UV-vis spectroscopy, and radioactive labeling, I discovered that WhiB3 is a 4Fe-4S cluster containing protein . This work was performed in collaboration with two EPR experts, Jack R Lancaster, Jr and Kevin E Redding. Furthermore, I showed that WhiB3 senses host signals such as nitric oxide (NO), oxygen (O2) and nutrients (sugars and fatty acids) via its redox active 4Fe-4S cluster. Since complex interactions of Mtb with the host environment underlies persistence of the pathogen as well as its virulence and sensitivity to drugs, the identification of WhiB3 as the first Mtb sensor of NO, O2 and nutrients represents a paradigm that impacts our current understanding of tuberculosis .
Since fatty acids are the main source of energy in vivo and also required by Mtb to synthesize virulence lipids, I hypothesized that WhiB3 maintains redox homeostasis by integrating fatty acid catabolism and lipid anabolism. Consistent with this view, I showed that WhiB3 regulates the production of several complex lipids (sulfolipids, cord factor, phthiocerol dimycocerosates) required for modulating the host immune response and thus virulence of Mtb. This work introduced a new concept in the redox biology of pathogens i.e. generation of intracytoplasmic reductive stress due to breakdown of highly reduced carbon sources (fatty acids) by Mtb during infection (Fig. 2). We suggested that mycobacterial persistence hinges on the strategies exploited by Mtb to successfully dissipate reductive stress during infection .
During my final year of post-doctoral training, I applied to Wellcome/DBT India Alliance intermediate fellowship. Because of my training in TB pathogenesis and redox biology, I developed a proposal on understanding the redox basis of dormancy and reactivation in TB. Receiving this prestigious grant allowed me to set up my research laboratory at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi. Importantly, Wellcome/DBT IA funding helped me to exploit my creative ideas in different fields. Last year, I was awarded a concept grant to exploit the role of redox stress during HIV infection by National Institute of Health (NIH) and International AIDS Society (IAS). Similarly, I received Innovative Young Biotechnologist Award (IYBA) by DBT, India in 2011 to develop a quantifiable system to measure redox stress in Mtb during infection. I enthusiastically look forward to continue my research on the fascinating areas of TB and TB-HIV pathogenesis.
1. Singh A, Gupta R, Vishwakarma RA, Narayanan PR, Paramasivan CN, et al. (2005) Requirement of the mymA operon for appropriate cell wall ultrastructure and persistence of Mycobacterium tuberculosis in the spleens of guinea pigs. J Bacteriol 187: 4173-4186.
2. Singh A, Mai D, Kumar A, Steyn AJ (2006) Dissecting virulence pathways of Mycobacterium tuberculosis through protein-protein association. Proc Natl Acad Sci U S A 103: 11346-11351.
3. Singh A, Guidry L, Narasimhulu KV, Mai D, Trombley J, et al. (2007) Mycobacterium tuberculosis WhiB3 responds to O2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival. Proc Natl Acad Sci U S A 104: 11562-11567.
4. Singh A, Crossman DK, Mai D, Guidry L, Voskuil MI, et al. (2009) Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response. PLoS Pathog 5: e1000545.