The exploitation of host autophagy and ubiquitin machinery by Mycobacterium tuberculosis in shaping immune responses and host defense during infection

Abstract

Intracellular pathogens have evolved various efficient molecular armaments to subvert innate defenses. Cellular ubiquitination, a normal physiological process to maintain homeostasis, is emerging one such exploited mechanism. Ubiquitin (Ub), a small protein modifier, is conjugated to diverse protein substrates to regulate many functions. Structurally diverse linkages of poly-Ub to target proteins allow enormous functional diversity with specificity being governed by evolutionarily conserved enzymes (E3-Ub ligases). The Ub-binding domain (UBD) and LC3-interacting region (LIR) are critical features of macroautophagy/autophagy receptors that recognize Ub-conjugated on protein substrates. Emerging evidence suggests that E3-Ub ligases unexpectedly protect against intracellular pathogens by tagging poly-Ub on their surfaces and targeting them to phagophores. Two E3-Ub ligases, PRKN and SMURF1, provide immunity against Mycobacterium tuberculosis (M. tb). Both enzymes conjugate K63 and K48-linked poly-Ub to M. tb for successful delivery to phagophores. Intriguingly, M. tb exploits virulence factors to effectively dampen host-directed autophagy utilizing diverse mechanisms. Autophagy receptors contain LIR-motifs that interact with conserved Atg8-family proteins to modulate phagophore biogenesis and fusion to the lysosome. Intracellular pathogens have evolved a vast repertoire of virulence effectors to subdue host-immunity via hijacking the host ubiquitination process. This review highlights the xenophagy-mediated clearance of M. tb involving host E3-Ub ligases and counter-strategy of autophagy inhibition by M. tb using virulence factors. The role of Ub-binding receptors and their mode of autophagy regulation is also explained. We also discuss the co-opting and utilization of the host Ub system by M. tb for its survival and virulence.Abbreviations: APC: anaphase promoting complex/cyclosome; ATG5: autophagy related 5; BCG: bacille Calmette-Guerin; C2: Ca²⁺-binding motif; CALCOCO2: calcium binding and coiled-coil domain 2; CUE: coupling of ubiquitin conjugation to ER degradation domains; DUB: deubiquitinating enzyme; GABARAP: GABA type A receptor-associated protein; HECT: homologous to the E6-AP carboxyl terminus; IBR: in-between-ring fingers; IFN: interferon; IL1B: interleukin 1 beta; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LGALS: galectin; LIR: LC3-interacting region; MAPK11/p38: mitogen-activated protein kinase 11; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK8/JNK: mitogen-activated protein kinase 8; MHC-II: major histocompatibility complex-II; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB1/p50: nuclear factor kappa B subunit 1; OPTN: optineurin; PB1: phox and bem 1; PE/PPE: proline-glutamic acid/proline-proline-glutamic acid; PknG: serine/threonine-protein kinase PknG; PRKN: parkin RBR E3 ubiquitin protein ligase; RBR: RING-in between RING; RING: really interesting new gene; RNF166: RING finger protein 166; ROS: reactive oxygen species; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; UBA: ubiquitin-associated; UBAN: ubiquitin-binding domain in ABIN proteins and NEMO; UBD: ubiquitin-binding domain; UBL: ubiquitin-like; ULK1: unc-51 like autophagy activating kinase 1.

Keywords: Autophagy; E3-Ub ligase; LC3; intracellular pathogens; phagolysosome; ubiquitin-binding receptors; virulence effectors; xenophagy.

Figure 1.

Diverse Ub-signals communicate different biological messages. (a) The diverse Ub-linkages coordinate different biological outcomes such as mono or multi-mono ubiquitination transmit signals for localization, control the activity of ubiquitinated substrates, proteolysis mediated by Ub-proteasome system, and autophagy. (b) K48 and K11 linkages usually transmit signals for degradation of short-lived folded proteins performed by 26S proteasome. K48-linked chains transmit signals for autophagic removal of invading pathogens and misfolded protein aggregates. K63-linked Ub chains transmit signals to remove aggregated proteins and participate in xenophagic clearance of invading cytosolic bacteria by autophagy. Diverse non-degradative processes are regulated by K63 linkages, including activation of DNA damage repair, assembly, and activation of signaling complexes. The main functions of K48- and K63-linked Ub chains are known. However, the crucial roles of newly discovered atypical linkages, including K6, K27, K29, and K33, are less known. Met1-linked linear chain takes part in removing invading pathogens, damaged mitochondria and activates the NFKB1 signaling pathway. (c) Small molecule modifiers of Ub such as acetylation, phosphorylation, and neddylation control various Ub functions. The functions of more complex hetero conjugated Ub are emerging now. S, substrate; Ub, ubiquitin; NEDD8, NEDD8 ubiquitin like modifier.

Figure 2.

The molecular players of the Ub-conjugation system. Ubiquitination is a three-step enzyme-catalyzed reaction coordinated by E1 or Ub-activating enzyme that catalyzes the ATP and Mg²⁺ dependent activation of Ub required to form a thioester bond in between catalytic cysteine on the E1 and C terminus glycine of Ub. The transfer of activated Ub from the Ub-conjugating enzyme (E2) to the substrate is mediated by the E3-Ub ligase. The removal and recycling of Ub from substrates are catalyzed by DUBs which maintain the Ub-pool. S, substrate; Ub, ubiquitin; DUB, deubiquitinase.

Figure 3.

E3-Ub ligase uses a distinct mechanism for the transfer of Ub to substrates. (a) E3-Ub ligase containing the RING domain transfers Ub to target protein substrates from E2-Ub conjugating complex. RING or similar UBOX containing E3-Ub ligases exist in multiple oligomeric states such as monomers, homodimers, and heterodimers. CUL3-E3 RING ligase comprises multiple subunits that coordinate interaction with the substrate. The complex contains adapter protein and substrate-interacting protein together with various CUL isoforms. The anaphase-promoting complex/cyclosome contains multiple subunits that coordinate interaction between target substrates and RING domain-containing E3-Ub ligase together with E2-Ub conjugating enzyme. The role and functions of multi-subunit complex E3-Ub ligases are emerging now. These complexes perform more complex hetero-conjugation of Ub to the substrates. (b and c) The HECT and RBR type E3-Ub ligases transfer Ub from E2-Ub conjugating enzymes to HECT or RING domain conserved cysteines followed by the Ub transfer to target substrates. S, substrate; Ub, ubiquitin; SR, substrate receptor.

Figure 4.

Structural features of PRKN and SMURF1 E3-Ub ligases which execute xenophagy mediated clearance of M. tb. (a) RING1 domain of PRKN consists binding site for Ub-conjugating E2 enzyme. RING2 domain of PRKN contains catalytic cysteine to form a covalent linkage with Ub. The other conserved domains, such as the UBL domain and REP linker region, sandwiched between the RING2 and IBR domains, inhibit RING1 binding to E2. The RING0 domain partially covers the catalytic cysteine residue present in the RING2 domain. (b) The N-terminal C2 domain binds phospholipids and plays an essential role in SMURF1 localization to the membrane. WW domains of SMURF1 are protein interaction domains required for binding with the targets. The transfer of Ub to the protein substrate is governed by the catalytically active C-terminal domain of the HECT family of E3-Ub ligases. The molecular size of both the E3-Ub ligases is shown. (C) PRKN and SMURF1 E3-Ub ligases provide immunity against M. tb. PRKN and SMURF1 ubiquitinate M. tb and its associated membranous structure in the cytosol to recruit autophagy receptors SQSTM1, CALCOCO2, and NBR1. The engagement of LC3 to the phagophore membrane and consequent fusion to the lysosome targets M. tb to xenophagy. REP, repressor element of PRKN.

Figure 5.

Structural features of xenophagy receptors. The xenophagy receptors (selective autophagy receptors), also known as sequestome-1-like receptors, include TAX1BP1, CALCOCO2, NBR1, SQSTM1, and OPTN. PB1 (dark pink), ZZ type zinc finger domains (light blue and dark blue), coiled-coil (purple), FW domain (light purple), LC3-interacting regions (LIRs) are marked by yellow. UBA domain (light pink) and SKICH domains are marked as light green. The size of the receptors is shown.

Figure 6.

M. tb co-opts the host Ub-system to manipulate and subdue host immunity. The surface immune receptors TLR2-TLR4 recognize M. tb or its associated molecular patterns to initiate downstream signaling events to generate innate immune responses against the pathogen. Downstream to TLRs, the adaptors TRAF6, TAB1-TAK1-binding protein 1, and TAB2-TAB3 sense Ub chains and affect the NFKB1 and MAP kinase signaling to produce proinflammatory cytokines TNF, IL12, IL6, and IL1B. The M. tb secreted effectors (e.g., Mpt53, PtpA, and Rv0222 co-opt the host Ub-system and affect the Ub-mediated activation of kinases MAP3K7 and TRAF6. It ultimately inhibits or activates the NFKB1 and MAPKs signaling and produces innate immune effector cytokines to dampen innate immunity against the pathogen. The host exploits one of the surface Ub-binding M. tb effectors [e.g., PE_PGRS29 (Rv1468c)] to start xenophagy against the pathogen and recruit receptor proteins SQSTM1, NBR1, CALCOCO2, and OPTN.

Figure 7.

M. tb uses its virulence effector proteins to inhibit autophagy. The M. tb proteins inhibit host-induced autophagy for its efficient intracellular survival inside macrophages. These proteins exhibit divergent mechanisms for the inhibition of autophagy. M. tb utilizes its effectors to dampen autophagy by inhibiting phagophore maturation, autophagosome fusion to the lysosome, and autolysosome acidification. The efficient mechanisms utilized by M. tb culminate in the reduction of autophagy flux. Exploring the interaction between these proteins will shed light on a better understanding of mechanisms of autophagy inhibition by M. tb.