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.

Prior to coronavirus disease 2019 (COVID-19), tuberculosis (TB) was the worst killer among infectious diseases. The union of these two obnoxious respiratory diseases can be devastating, with severe public health implications. The COVID-19 pandemic has affected all TB-elimination programmes due to the severe burden on healthcare systems and the diversion of funds and attention towards controlling the pandemic. The emerging data show that the COVID-19 pandemic caused a marked decrease in case notifications and bacille Calmette–Guérin immunisations, ultimately promoting disease transmission and increasing the susceptible population. The similarity between the clinical characteristics of TB and COVID-19 adds to the public health complications, with evidence of immune dysregulation in both cases leading to severe consequences. Clinical evidence suggests that severe acute respiratory syndrome coronavirus 2 infection predisposes patients to TB infection or may lead to reactivation of latent disease. Similarly, underlying TB disease can worsen COVID-19. Treatment options are limited in COVID-19; therefore, using immunosuppressive and immunomodulatory regimens that can modulate the concomitant bacterial infection and interaction with anti-TB drugs requires caution. Thus, considering the synergistic impact of these two respiratory diseases, it is crucial to manage both diseases to combat the syndemic of TB and COVID-19. M. tuberculosis is a deadly human pathogen, and the emergence of COVID-19 has hampered TB control measures. Better co-infection management can prevent disease-related complications and produce favourable outcomes.https://bit.ly/3LiiV57

Reductive evolution has endowedMycobacterium tuberculosis(M. tb) with moonlighting in protein functions. We demonstrate that RipA (Rv1477), a peptidoglycan hydrolase, activates the NFκB signaling pathway and elicits the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12, through the activation of an innate immune-receptor, toll-like receptor (TLR)4. RipA also induces an enhanced expression of macrophage activation markers MHC-II, CD80, and CD86, suggestive of M1 polarization. RipA harbors LC3 (Microtubule-associated protein 1A/1B-light chain 3) motifs known to be involved in autophagy regulation and indeed alters the levels of autophagy markers LC3BII and P62/SQSTM1 (Sequestosome-1), along with an increase in the ratio of P62/Beclin1, a hallmark of autophagy inhibition. The use of pharmacological agents, rapamycin and bafilomycin A1, reveals that RipA activates PI3K-AKT-mTORC1 signaling cascade that ultimately culminates in the inhibition of autophagy initiating kinase ULK1 (Unc-51 like autophagy activating kinase). This inhibition of autophagy translates into efficient intracellular survival, within macrophages, of recombinantMycobacterium smegmatisexpressingM. tbRipA. RipA, which also localizes into mitochondria, inhibits the production of oxidative phosphorylation enzymes to promote a Warburg-like phenotype in macrophages that favors bacterial replication. Furthermore, RipA also inhibited caspase-dependent programed cell death in macrophages, thus hindering an efficient innate antibacterial response. Collectively, our results highlight the role of an endopeptidase to create a permissive replication niche in host cells by inducing the repression of autophagy and apoptosis, along with metabolic reprogramming, and pointing to the role of RipA in disease pathogenesis.

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 thatM. tbPE6 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-α, 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 apoptosisviaincreased 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, eIF2α, IRE1α, 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 reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. RecombinantMycobacterium smegmatis(M. smegmatis) containingpe6displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host–pathogen interaction duringM. tb infection.

TRIM proteins are characterized by their conserved N-terminal RING, B-box, and coiled-coil domains. These proteins are efficient regulators of autophagy, apoptosis, and innate immune responses and confer immunity against viruses and bacteria. TRIMs function as receptors or scaffold proteins that target substrates for autophagy-mediated degradation. Most TRIMs interact with the BECN1-ULK1 complex to form TRIMosomes, thereby efficiently targeting substrates to autophagosomes. They regulate the functions of ATG proteins through physical interactions or ubiquitination. TRIMs affect the lipidation of MAP1LC3B1 to form MAP1LC3B2, which is a prerequisite for phagophore and autophagosome formation. In addition, they regulate MTOR kinase and TFEB, thereby regulating the expression of ATG genes. TRIM proteins are efficient regulators of apoptosis and are crucial for regulating cell proliferation and tumor formation. Many TRIM proteins regulate intrinsic and extrinsic apoptosis via the cell surface receptors TGFBR2, TNFRSF1A, and FAS. Mitochondria modulate the anti- and proapoptotic functions of BCL2, BAX, BAK1, and CYCS. These proteins use a multipronged approach to regulate the intrinsic and extrinsic apoptotic pathways, culminating in coordinated activation or inhibition of the initiator and executor CASPs. Furthermore, TRIMs can have a dual effect in determining cell fate and are therefore crucial for cellular homeostasis. In this review, we discuss mechanistic insights into the role of TRIM proteins in regulating autophagy and apoptosis, which can be used to better understand cellular physiology. These findings can be used to develop therapeutic interventions to prevent or treat multiple genetic and infectious diseases.Graphical Abstract

Permeation through bacterial cells for exchange or uptake of biomolecules and ions invariably depend upon the existence of pore-forming proteins (porins) in their outer membrane. Mycobacterium tuberculosis (M. tb) harbours one of the most rigid cell envelopes across bacterial genera and is devoid of the classical porins for solute transport across the cell membrane. Though canonical porins are incompatible with the evolution of permeability barrier, porin like activity has been reported from membrane preparations of pathogenic mycobacteria. This suggests a sophisticated transport mechanism that has been elusive until now, along with the protein family responsible for it. Recent evidence suggests that these slow-growing mycobacteria have co-opted some of PE/PPE family proteins as molecular transport channels, in place of porins, to facilitate uptake of nutrients required to thrive in the restrictive host environment. These reports advocate that PE/PPE proteins, due to their structural ability, have a potential role in importing small molecules to the cell's interior. This mechanism unveils how a successful pathogen overcomes its restrictive membrane's transport limitations for selective uptake of nutrients. If extrapolated to have a role in drug transport, these channels could help understand the emergence of drug resistance. Further, as these proteins are associated with the export of virulence factors, they can be exploited as novel drug targets. There remains, however, an interesting question that as the PE/PPE proteins can allow the 'import' of molecules from outside the cell, is the reverse transport also possible across the M. tb membrane. In this review, we have discussed recent evidence supporting PE/PPE's role as a specific transport channel for selective uptake of small molecule nutrients and, as possible molecular export machinery of M. tb. This newly discovered role as transmembrane channels demands further research on this enigmatic family of proteins to comprehend the pathomechanism of this very smart pathogen.