Autophagy induction by Mycobacterium indicus pranii promotes Mycobacterium tuberculosis clearance from RAW 264.7 macrophages

December Autophagy induction by Mycobacterium indicus pranii promotes Mycobacterium tuberculosis clearance from RAW 264.7 macrophages Bindu Singh 0 1 Mohd Saqib 0 1 Ananya Gupta 0 1 Pawan Kumar 1 Sangeeta Bhaskar 0 1 0 Product Development Cell, National Institute of Immunology , New Delhi, Delhi , India , 2 Special Centre for Molecular Medicine, Jawaharlal Nehru University , New Delhi , India 1 Editor: Seyed Ehtesham Hasnain, Indian Institute of Technology Delhi , INDIA Mycobacterium indicus pranii (MIP) is a potent vaccine candidate against tuberculosis (TB) as it has demonstrated significant protection in animal models of tuberculosis as well as in clinical trials. Higher protective efficacy of MIP against TB as compared to BCG provoked the efforts to gain insight into the molecular mechanisms underlying MIP mediated protection against Mycobacterium tuberculosis (M.tb). Autophagy, initially described as a cell survival mechanism during starvation, also plays a key role in host resistance to M.tb. Virulent mycobacteria like M.tb, suppresses host autophagy response to increase its survival in macrophages. Since mycobacterial species have been shown to vary widely in their autophagyinducing properties, in the present study, we examined the autophagy inducing efficacy of MIP and its role in MIP-mediated protection against M.tb. MIP was found to be potent inducer of autophagy in macrophages. Induced autophagy was responsible for reversal of the phagosome maturation block and phagolysosome fusion inhibition in M.tb infected macrophages, which ultimately lead to significantly enhanced clearance of M.tb from the macrophages. This is an important study which further delineated the underlying mechanisms for significant immunotherapeutic activity observed in TB patients / animal models of tuberculosis, given MIP therapy along with chemotherapy. - Copyright: © 2017 Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Core research grant of National Institute of Immunology. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Tuberculosis (TB), a chronic infectious disease, remains one of the world's deadliest communicable diseases (WHO report 2016). Mycobacterium tuberculosis (M.tb), the causative organism of TB, is a highly successful pathogen as it has evolved number of survival strategies to evade the host immune mechanisms [ 1 ]. Blocking of phagosome maturation and phago-lysosome fusion, interference with antigen presentation, resistance to reactive oxygen and nitrogen intermediates [ 2,3 ], alteration of host cell apoptotic pathways [4] and inhibition of autophagy in host cells [ 5 ] are some of the strategies which enhance M.tb survival inside the macrophages. Abbreviations: 3-MA, 3-Methyl Adenine; CFU, Colony forming units; DAPI, 40,6-Diamidine-20phenylindole dihydrochloride; DQ-BSA, Dyequenched Bovine Serum Albumin; EDTA, Ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; LC3, Microtubule-associated proteins 1A/1B light chain 3B; M.tb, Mycobacterium tuberculosis; MIP, Mycobacterium indicus pranii; MOI, Multiplicity of infection; PBS, Phosphate buffered saline; PFA, Paraformaldehyde; PVDF, Polyvinylidene difluoride; RPMI, Roswell Park Memorial Institute medium; siRNA, Small interfering RNA; TB, Tuberculosis. Autophagy, a lysosomal degradation pathway which contributes to maintenance of intracellular homeostasis, has been shown to be an integral part of both adaptive and innate immunity [ 6 ]. It was initially known as stress response involved in cell survival during nutrient starvation condition and for its role in maintaining intracellular homeostasis, by eliminating surplus or damaged organelles and degradation of superfluous, misfolded and damaged proteins [ 7 ]. Subsequent studies showed the innate defence role of autophagy against invading pathogens including M.tb [8±10]. One of the important survival strategies of M.tb is to evade acid hydrolases by inhibiting phagosome-lysosome fusion [ 11,12 ]. Autophagy induction by physiological, pharmacological or immunological means was found to reduce intracellular M.tb survival by targeting it for lysosomal degradation [13]. The importance of autophagy genes in restricting intracellular growth of M.tb was confirmed by genome-wide small interfering RNA (siRNA) screenings in which 74 target genes were knocked down using target-specific siRNAs and their effect on M.tb survival was tested. (...truncated)