CDFD :: Molecular Cell Biology

Broad Research Area:

Molecular dissection of the changes in innate and adaptive immune responses signaling networks of the host by invading pathogens and/or stress.

The activation of macrophage leads to production of a wide range of inducible gene products that are essential in developing innate and subsequent acquired immune phenotypes like T helper type 1 (Th1) or Th2 responses. The major goals of our laboratory encompass from understand ing the cell signaling networks that are important for macrophage activation to elucidating how expression of various genes are modulated during various pathophysiological conditions leading to establishment of diseased states using state of the art molecular biology procedures like recombinant DNA technology, yeast two hybrid system, proteomics and immunological techniques. In particular, we are looking at the immunomodulation during tuberculosis and stress. These studies are intended to develop novel immunomodulatory drugs with therapeutic potential against tuberculosis and other health problems like ageing and cancer. Our main research interests include,

The free radicals like nitric oxide (NO) and reactive oxygen species (ROS) and their by-products cause oxidative damage of cellular components like lipids, proteins, DNA, and eventually leads to cell death. Despite the well known activity of NO/ROS in antibody independent cellular cytotoxicity, the effect of excess NO/ROS in modulating the homeostasis of the immune system is not well documented. Our studies for the first time provide evidence that, free radicals can act as secondary signaling cues that can modulate macrophage effector functions and adaptive immune response of the host. We have demonstrated that these molecules could target the calmodulin-c-rel signaling cascades to subsequently inhibit interleukin-12 (IL-12) induction in activated macrophages. Production of free radicals is known to be a primary defensive mechanism elicited by the host because of their potent cytotoxicity on pathogens and considered to be an important component of the innate immunity. However, our work clearly highlights that excess free radicals produced under certain pathophysiological conditions actually can surpass their beneficial role to cause immunosuppression thus preventing desirable forward amplification of the defensive immune responses and may favor survival of intracellular pathogens and/or cause other problems like ageing and neoplasia.

We are currently studying alterations of macrophage effector and APC functions by NO/ROS and cell signaling pathways involved in such modulations. Also we are studying the immunore-gulatory role of NO/ROS during stress and infection (tuberculosis). Since NO/ROS cause for senescence associated losses in physiological functions including the immune system, a research in this area is likely to help us designing effective interventions for improving deteriorated immune functions in elderly people or people exposed to chronic oxidative stress during infection (tuberculosis).

Tuberculosis (TB) is declared a global emergency by the WHO, the first disease to be so desig-nated which kills about 3 million people a year. The bacillus Mycobacterium tuberculosis re-mained the leading cause of mortality in the world, even when we are entering into the new millennium of technical and medical excellence. An extremely resilient cell wall, very slow growth, highly contagious nature and a number of cunning strategies adopted by the pathogen to evade host immune attack make it one of the least tractable organisms to work with. The rapid spread of tuberculosis in AIDS patients and emergence of multidrug resistant strains renders the situation still grave. The bacterium has a very sophisticated signal transduction systems and potentialities to adapt into a variety of hostile environments. There have been extensive attempts worldwide to identify the mycobacterial components that have an important role in the establishment of the infection. But till date only little information is available regarding the molecular basis of mycobacterial virulence. Development of vaccines is getting hampered because of the facts that the host-bacilli interactions and molecular basis of pathogen invasion are not well understood yet. Identification and functional characterization of the set of genes expressed by the M. tuberculosis bacilli during interaction with macrophage will be helpful to increase our understanding of pathogenic mechanisms that result in disease and may provide insights into potential vaccine strategies and novel drug targets.

The macrophage mediates the first line of defense in the host. The M. tuberculosis bacilli play various strategies to suppress the macrophage-innate effector and APC (antigen presenting cell) functions and use the host's defense weapon as its comfortable home. In various projects we are approaching to identify and characterize the mycobacterial ORFs/proteins that enable the bacilli to survive and multiply within the macrophages. We also approach to identify the down-stream signal transduction pathways in macrophages modulated during TB infection, which is important to understand how the bacterium invades the macrophage cells successfully and this work will provide new insights about the detrimental endogenous immune responses triggered by the mycobacterium bacilli and shed some light on the molecular basis of pathogenesis of this dreadful pathogen. We have demonstrated that some of the candidate PE/PPE family proteins are involved in the pathogenesis of tuberculosis (filed USA Patent-1, 2008) and thus constitute potent drug targets.

In the area of tuberculosis diagnosis, it is important to identify suitable antigens that can differentiate active tuberculosis patients from BCG-vaccinated individuals. Since many PE/PPE proteins are present only in the pathogenic mycobacteria like M. tuberculosis and few of these are found to be overexpressed in macrophages during infection, it is possible that these proteins are highly sensitive to detect patients with active tuberculosis. We have identified one candidate protein which displays stronger and specific immunoreactivity against the sera obtained from clinically active TB patients compared to PPD, ESAT-6 and hsp60 and could differentiate TB-patients from the BCG-vaccinated controls (filed USA Patent-2, 2008). We are interested to identify potent markers that can be used to differentiate active tuberculosis patients from BCG-vaccinated individuals since currently used Mantoux test cannot distinguish such differences reliably.

Research Contribution

Since joining CDFD, Hyderabad as Staff Scientist III, the major focus of research of Dr. Sangita Mukhopadhyay have been revolving around infection biology and immunity. Dr. Sangita Mukhopadhyay made outstanding scientific advancements in field of tuberculosis (TB) research. She has established research collaborations with several hospitals/Medical Institutes which yielded excellent contributions in the field of Tuberculosis and Inflammation disease biology. Her research has been pivotal in understanding the molecular mechanisms of host-pathogen interactions with respect to tuberculosis, designing of immunomodulators which has significant potentials for improving human health.

Despite lacking classical toxins, a smart pathogen like Mycobacterium tuberculosis (Mtb) employs a diverse array of spatio-temporally regulated virulent factors to evade host’s protective immune responses t to ensure its survival and pathogenesis. Therefore, understanding the molecular mechanisms by which mycobacterial virulence factors help to establish Tuberculosis is crucial in order to design effective therapeutics against M. tuberculosis. The seminal works carried out by Dr. Mukhopadhyay threw considerable lights for the first time on how the innate and the adaptive immune responses of host are hijacked by M. tuberculosis through the virulent proteins like ESAT-6, PknG and some members of the intriguing PE/PPE family proteins (PE11, PPE2 and PPE18) which could be potential drug targets (Figure 1). She has shown that ESAT-6 protein inhibits class-I antigen presentation and intracellular iron homeostasis by physically interacting with beta-2 microglobulin (β2M) (PLoS Pathog.[2014]10:e1004446, J. Immunol.[2020]205:3095; J. Immunol.[2019]203:1918). Again for the first time she showed that Mtb PPE18 protein inhibits the protective Th1/pro-inflammatory responses and MHC class-II antigen presentation by masking part of the TLR2-ectodomain (J. Immunol.[2009]183:6269; J. Immunol.[2011]186:5413; J. Immunol.[2016]197:1776; Eur J Immunol[2021] 51:603-619). Based on these findings, we demonstrated that TLR2-LRR 11~15 domain as well as ESAT-6:β2M can be novel targets for host-directed therapy and designed two FDA approved drugs. Also, She found that PPE2 protein mimics eukaryotic transcription factor to translocate to nucleus where it sterically inhibits transcription from iNOS promoter (Sci. Rep.[2017]7:39706). In addition, the protein has SH3 domain that allows it to interact with p67phox resulting in inhibition of reactive oxygen species (J. Immunol.[2019]203:1218) and also mast cells and myeloid haematopoiesis (Immunobiology[2021]226:152051; EMBO Mol Med[2022]14(9):e14891) and thus, is responsible to suppress innate defense response of host which are crucial for anti-Mtb immunity (J. Immunol.[2021]207:2393; FEBS J[2022]289:4146-4171]). She is now looking at role of PPE2 in host-pathogen interaction and disease pathogenesis in TB. Again, for the first time we showed that Mtb PE11 protein with esterase activity plays a crucial role in regulating bacterial cell wall architecture and provide defense to the bacilli against antibiotics and cellular stressors (Sci. Rep.[2016]6:21624). She is now investigating in detail the role of PE11 in modulating host responses. Also, for the first time, she showed Mtb PknG protein targets Rab7l1-signaling to block phagosome-lysosome fusion for favoring Mtb survival inside macrophages and Rab7l1-PknG as a crucial drug target in TB (J. Immunol.[2018]201:1421; Immunology[2022]165:328). Also she has undertaken research to establish that excess free radicals produced during infection can cause immunosuppression and therapeutic effect of anti-oxidant to boost the immune response against tuberculosis ( Blood[2006]; J. Immunol.[2010]). Thus, her research ushers promising approaches for host directed immune therapy targeting ESAT-6, PPE2 and PPE18 proteins, identification of repurposed drugs and application of anti-oxidant (N-acetyl cysteine) to increase host protective responses against TB. The study will make a long-term contribution in preventing infection and disease in countries with high burdens of TB. Also, she is involved in understanding the causal factors and mechanism of TB-associated pathophysiological disorders and designing of suitable therapeutics to ameliorate these health problems.

Further, Dr. Mukhopadhyay translated her original research into therapeutic applications leading to filing of several patents (Indian patent[2016]; J. Immunol.[2018]200:3587; Indian Patent[2019]; USPTO[2020]). She is now exploring therapeutic application in wound healing as well as treating inflammatory bowel disease and melanoma cancer which are associated with extreme inflammation. In the field of ‘Disease Biology’, She demonstrated a strong focus on innovation and adaptation and as a result filed several Indian and USA patents and published 71 papers in peer reviewed International Journals of high impact factor. In this direction of research, she have been bestowed with many highly prestigious ‘National Awards’ and the TATA Innovation Fellowship, 2017-2018 of DBT and J. C. Bose Fellowship and continuous funding supports from various funding agencies like TWAS, Italy, DBT, Govt of India, DST-SERB, Govt of India, CSIR, Govt of India and ICMR, Govt of India. I am also involved for commercialization of our innovations through Industry-Academy collaboration with NC TRAC and BCIL. Our research has been highlighted in the Hindu and vigyanprasar Government Web portal (https://www.thehindu.com/sci-tech/health/novel-mechanism-may-lead-to-better-tb-control/article6764393.ece and http://vigyanprasar.gov.in/isw/A-new-boost-to-anti-tb-crusade.html). Her contributions to original scientific research has been well recognized by the prestigious ASH, USA and AAI, USA by electing her as a member and also elected as Fellow of ‘The National Academy of Sciences, India; Indian Academy of Sciences, Bangalore; Indian National Science Academy, New Delhi and Telangana Academy of Sciences, Telangana.. She is also a member of the various Task force committees of various national funding agencies like CSIR, DST-SERB, ICMR, DBT (till 2018). She is serving as an expert member in various committees of National Importance like INSA, DST-INSPIRE, CSIR-SPMF, DST-WOS (till 2019); IoE of University of Hyderabad, UoH Board of studies, SAC of NIBMG and NCCS. Also, she is an active committee member of the Governing body of LVPEI, Hyderabad.