Mycobacterium tuberculosis Protein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival Neha Sharma 1,2† , Mohd Shariq 1† , Neha Quadir 1,2† , Jasdeep Singh 2 , Javaid A. Sheikh 3 , Seyed E. Hasnain 4,5 * and Nasreen Z. Ehtesham 1 * 1 Indian Council of Medical Research-National Institute of Pathology, New Delhi, India, 2 Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India, 3 Department of Biotechnology, School of Chemical and Life Science, Jamia Hamdard, New Delhi, India, 4 Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi, India, 5 Department of Life Science, School of Basic Science and Research, Sharda University, Greater Noida, India Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-a, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2a, IRE1a, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the Frontiers in Immunology | www.frontiersin.org July 2021 | Volume 12 | Article 696491 1 Edited by: Samantha Leigh Sampson, Stellenbosch University, South Africa Reviewed by: Suraj Sable, Centers for Disease Control and Prevention (CDC), United States Yunhao Tan, AbbVie (United States), United States *Correspondence: Seyed E. Hasnain seyedhasnain@gmail.com; seyed.hasnain@sharda.ac.in; seh@dbeb.iitd.ac.in Nasreen Z. Ehtesham nzehtesham@gmail.com † These authors have contributed equally to this work Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology Received: 16 April 2021 Accepted: 21 June 2021 Published: 12 July 2021 Citation: Sharma N, Shariq M, Quadir N, Singh J, Sheikh JA, Hasnain SE and Ehtesham NZ (2021) Mycobacterium tuberculosis Protein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival. Front. Immunol. 12:696491. doi: 10.3389/fimmu.2021.696491 ORIGINAL RESEARCH published: 12 July 2021 doi: 10.3389/fimmu.2021.696491