Kumar D, Nath L, Kamal MA, Varshney A, Jain A, Singh S, Rao KV. Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis. Cell. 2010 Mar 5;140(5):731-43.

Host factors responsible for the persistence of Mycobacterium tuberculosis revealed

Indian scientists have achieve­d an important breakthrough in the fight against tuberculosis with researchers claiming to have uncovered hitherto unknown host factors which aid in the survival and persistence of Mycobacterium tuberculosis (Mtb) after it infects man. Dr. K. V. S. Rao and his group from International Centre for Genetic Engineering and Biotechnology, New Delhi, employed an exhaustive genome wide short interfering RNA (siRNA) screen to identify 275 molecules crucial for the bacterium, associated in a dense network with each other. The study, published in March 2010 issue of the prestigious journal “Cell” sheds light on modules which are either engaged or perturbed after infection by Mtb.

siRNA screen has emerged as a valuable tool to study loss-of-function phenotype of a gene. The generation of large-scale mammalian siRNA libraries during last few years has provided a powerful platform to carry out phenotype-based large-scale genome-wide screen in mammalian cells. This has allowed scientists to effectively demarcate biomolecular networks responsible for various cellular processes, as has been achieved in this study.

Dr. Rao’s team employed human whole-genome siRNA library to target a total of 18,174 genes, as a mixture of four siRNAs per target gene. This was done in human macrophage-like THP-1 cells, which were first infected with Mycobacterium tuberculosis virulent strain H37Rv.

Giving details about this work, Dr. Rao said, “The question of how pathogen diversification influences intracellular adaptation has never been addressed for any infection. It provides a better understanding of the equilibration process that the pathogen adopts within the intracellular milieu of the host cell. Several of the targets identified also serve as targets for drug development”.

Since the work was published, the team has already developed potent inhibitors against some of the targets in collaboration with a Pharma company. These inhibitors can substantially reduce the mycobacterial load. Morever, these inhibitors have been found to be equally effective against drug-sensitive, multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of TB in vitro as well as in mouse models,  when subsequent investigations were conducted by the team. If these inhibitors happen to reach clinical trials, they might help treat the drug resistant TB cases, and thus considerably reduce overall disease burden.

The technique of siRNA screening is not an easy one. Talking about the challenges the team faced, Dr. Rao remarked, “Performing the screen itself was challenging, particularly since it was done manually. That is, we recruited 18 summer students, trained them, and then put them in an assembly line format for completing the screen. This is the first time anywhere where a genome wide siRNA screen has been done manually.”

Further, the team also performed a subsequent screen against a diverse panel of field isolates. This exercise revealed that the molecular composition of the host interface varied with both the genotype and the phenotypic properties of the pathogen. Analysis of these differences led to identification of host factors which were invariantly involved in the survival of Mtb, regardless of the diversification in adaptive mechanisms employed by it.

Taking their studies a step ahead, the group also integrated the data obtained by siRNA screen with time series microarray data to transform it into a time-resolved perspective, which again is a first-of-its-kind.

“At one level we are adopting a systems level approach to understand the dynamics of how Mtb equilibrates within the host cell. In addition, we are also aggressively pursuing the drug development angle”, says Dr. Rao.

Tuberculosis in this part of the world is a major health problem leading to death and debility in a large chunk of population. These findings come at a time when the estimated incidence of TB in India amounts to 1.8 million new cases annually. A growing economy can ill afford to lose its human resource to a scourge so threatening. With various strains of Mtb infecting humans, concerted efforts are required to decipher strain-specific host-pathogen interaction factors involved in pathogen survival.

The standard treatment of TB with isoniazid, rifampicin, pyrazinamide and ethambutol is beset with the occurrence of a relapse rate of 2-3%, drug resistance, thrombocytopenia, rashes, itching, fever, hepatitis etc. Whether using chemogenomics approaches to perturb the regulatory networks deciphered in this study will lead to the development of novel bactericidal agents in the fight against tuberculosis, only time will tell.

This study also has wider implications in systems biology of the host-pathogen interactions. Biologists can now use similar methodologies to further understand host-pathogen co-evolution of the intracellular pathogens like Mycobacterium, Leishmania and Plasmodium. These unicellular pathogens are clever co-evolvers and past-masters in modulating the host’s environment for their survival and persistence. In some types, the host’s immune system cells themselves are used to gain entry into the host (à  la Trojan horse). It’s quite baffling how these pathogens befool the host after initial infection.  There is now hope of answering such tricky questions pertaining to the biology of intracellular pathogens by adopting a systems approach, such as this one.

- Puneet Raina

PR holds a Master's in Zoology and is currently pursuing his PhD on the molecular biology of programmed cell death in Leishmania donovani at Panjab University, Chandigarh. He is a recipient of both Junior and Senior Research Fellowships from CSIR. He was also awarded Science Talent Promotion Scholarship by the Department of Science and Technology, J&K in 1994. His research interests include molecular parasitology, immune response and cell-signaling, programmed cell death, gene knockdown, antisense oligonucleotide design, & systems biology of host-parasite interactions. He has undertaken temporary teaching assignments (undergrad and postgraduate classes) at Panjab University. Apart from research, he likes writing science articles. This is his first news feature for IndiaBioscience.