Thsti - Translational Health Science and Technology Institute

Posdoctoral Fellow, University of Massachusetts Medical School, Worcester, Massachusetts, USA
Posdoctoral Fellow, University of Nebraska-Lincoln, Nebraska, USA
Ph.D. Indian Veterinary Research Institute, Izatnagar, U.P., India
M.Sc. (Animal Biotechnology), National Dairy Research Institute, Karnal, India
B.V.Sc and A.H, Orissa University of Agriculture and Technology, Bhubaneswar, India

I. Understanding the biology of Mycobacterial Persistence

Globally, a third of human population is infected with Mycobacterium tuberculosis(Mtb), the causative agent of tuberculosis. Mtb, being an obligate intracellular pathogen, has lived to co-evolve with human for centuries. Unlike actively infected individuals, latently infected population harbors pathogen for decades without showing any overt symptoms. This interesting phenotype is attributed to a slow growing, metabolically altered sub set of heterogeneous Mtb population called ‘persisters’. These persisters are refractory to anti-mycobacterial insult and can only be targeted by a strict regimen comprising of a combination of drugs for an unusually extended period. The protracted regimen triggers non-compliance and contributes towards increase in the frequency of cases involving MDR and XDR tuberculosis.

A. Bacterial metabolism, physiology and persistence

The ability of the pathogen to “adapt” to host environment is key to its persistence. Understanding bacterial metabolism and physiology is important to decipher the mechanism and pathways critical for persistence of mycobacteria inside host. Since Mtb is an obligate intracellular pathogen, it depends on host for its nutritional requirements. Inside the host, Mtb subsists mainly on host derived fatty acid and cholesterol as a preferred carbon source. Although Mtb ingests cholesterol throughout the infection process, cholesterol becomes essential only during the later stage of chronic infection. Genetic and molecular understanding of cholesterol utilization, its mechanism and relevance would contribute significantly in designing novel intervention strategies in the treatment of tuberculosis. The knowledge acquired on the genes involved in uptake and metabolism of cholesterol in Mtb is very likely to generate new and more efficient drug targets. The role of cholesterol metabolism in mycobacterial persistence would also be better understood. The ultimate goal will be to generate an interactome map of the regulatory pathways of cholesterol utilization in Mtb.

B. Cholesterol catabolic pathways as therapeutics target

Current tuberculosis treatment regimen involves multiple drugs for a prolong period. The duration could be from three months to two years depending on the type of infection. Prolong treatment leads to non-compliance and emergence of newer drug resistance strains. Shortening the therapy would go a long way in alleviating this problem. It is widely perceived that the major culprits are the so-called non-replicating and metabolically inactive “persister” population. The importance of cholesterol metabolism during the persistence stage of Mtb infection and its potential role in generation of persisters is very intriguing. In light of the above facts and hypothesis the focus of the current proposal is to screen for chemical inhibitors that specifically target these pathways. The long-range goal would be identify novel anti-tubercular drugs that specifically targets “persisters”. These novel compounds in combination with the standard frontline anti-tubercular drugs would significantly enhance the success rate in tuberculosis therapy.

C. Iron regulation in Mtb and its implications on mycobacterial persistence

Although, iron is essential for most of the bacteria, excess of intracellular free iron is toxic. Failure to do so might lead to death either due to iron deficiency or toxicity. Hence, the acquisition and storage of iron in bacteria is tightly regulated. Since iron deprivation is also one of the anti-microbial strategies that the host adopts, both pathogen and the host compete for the limited iron during infection. We for the first time have demonstrated that the Mtb transcription repressor protein sufR_TBregulates the ISC operon and has a role in controlling the intracellular iron homeostasis in Mtb. Disruption of the iron homeostasis in ΔsufR_TBdecreased the fitness of the mutant strain to grow inside mouse bone marrow-derived macrophages. The transcription repressor protein sufR_TBwas also required for growth of Mtb under oxidative and nitrosative stress conditions. The enhanced biofilm production phenotype observed in ΔsufR_TBis intriguing and suggests a role of intracellular iron homeostasis in the generation of biofilms in mycobacteria. Finally, we demonstrated that the sufR_TBprotein mediated regulation of Fe homeostasis in Mtb is required for Mtb to persist inside the host.

D. Screening and Identifying new molecular scaffolds targeting persisters

We in our lab have successfully identified proteins critical for the generation and maintenance of mycobacterial persisters. The aim of the proposal is to shorten the treatment regimen by identifying novel compounds that specifically target these proteins thereby significantly decreasing the frequency of generation of persisters during Mtb infection.

II. Live recombinant M bovisBCG (rBCG) strain as a better vaccine candidate against tuberculosis

Although, Mycobacterium bovis derived BCG is widely been used as vaccine to prevent tuberculosis, its effectiveness towards controlling tuberculosis is questionable. BCG is shown to be good at controlling severe forms of tuberculosis in infants, but fails to do so in case of pulmonary tuberculosis in adults. The challenge is to design a vaccine strain that is a) safe, b) immunogenic, c) longlasting and d) protects against tuberculosis caused by all strains.We in our lab are working on strategies to generate rBCG strains that we believe will be more immunogenic and hence would generate better and long lasting protective immune response against Mtb.

III. Host-Directed Therapies against tuberculosis

We are also interested in identifying and targeting critical host proteins that facilitates mycobacterial survival inside host. We are currently developing protocols for CRISPR-cas9 based strategies to delete specific genes for development of knockout cell lines. The plan is to design an unbiased approach towards screening and identifying host genes that modulate growth of Mtb inside cells. The long-term goal is to identify inhibitors against these genes to be used as drug against tuberculosis.

DR.AMIT KUMAR PANDEY

Ph.D.

ASSOCIATE PROFESSOR

Publication