Mycobacterial infection alters host mitochondrial activity

Mitochondria are the power center of the cell, which are responsible not only for ATP synthesis but the only source for crucial metabolites, signaling molecules, and the house of innate immune signaling. They are double membraned structures with a porous outer membrane that allows passive diffusion of molecules (<5 kDa) and a densely folded inner membrane that is rigid owing to the protein cardiolipin. The outer mitochondrial membrane is a platform for many cell signaling processes and harbors several carrier proteins. The intermembrane space is a home for several damage-associated molecular patterns (DAMPs), whose release into the cytosol is recognized as a signal for cell death (Fig. 1). The ridged inner membrane carries the respiratory electron transport complexes. The inner membrane surrounds the mitochondrial matrix that harbors mtDNA and ribosomes (for further details please refer to Lobet et al., 2015). Mitochondria are usually elongated with internal cristae that are demarcated. The mitochondria are highly dynamic and physically connected to other subcellular organelles at particular sites referred to as MICOS (Mukherjee et al., 2021; Stephan et al., 2020), which are the sites for the dynamic exchange of matter and information. The interorganellar interaction is mediated by specific proteins for each organelle. For instance, Mfn2 for ER, Plin5 for lipid droplets, and vCLAMP for vacuole (reviewed in (Rambold and Pearce, 2018). The loss of inter-organelle communication results in dysfunctional mitochondria (Xia et al., 2019) that affect organellar dynamics, Ca2+ signaling, lipid homeostasis, and immune response leading to various disease states (Murley and Nunnari, 2016; Tubbs and Rieusset, 2017; West et al., 2011).

The classical function associated with mitochondria is the aerobic generation of energy. It occurs in two steps, first, the conversion of pyruvate to oxalate, which produces three NADH and one FADH2 through a series of biochemical conversions (TCA cycle), and second, their oxidative phosphorylation through a series of electron transport chain proteins, producing 36 ATP (OXPHOS) and is more efficient than its anaerobic counterpart, glycolysis.

Mitochondria is the seat for the TCA cycle, urea cycle, β-oxidation of fatty acids that churns out numerous intermediates metabolites that could not only be directly consumed or converted to other forms for macromolecule biosynthesis (including amino acids, nucleotide biosynthesis, fatty acid biosynthesis) but also influence cell fate and function through chromatin modifications and DNA methylation (reviewed in (Martínez-Reyes and Chandel, 2020)). These metabolites additionally serve in redox homeostasis and metabolic waste management (Spinelli and Haigis, 2018).

The mitochondria respond to diverse environmental cues dynamically by signaling mediators that invariably reach mitochondria. Subsequently, a response is generated by crosstalk with other subcellular organelles (Bussi and Gutierrez, 2019). Besides, mitochondria are a source of signaling molecules such as ROS, which are also transferred to other sites of action, such as phagosomes. Mitochondrial damage releases molecules such as mtDNA, ROS, succinate, ATP, N-formyl peptides, cytochrome C and TFAM (Mitochondrial transcription factor A), known as mitochondrial Damage Associated Molecular Patterns (DAMPs). These DAMPs are recognized by various PRRs of the host, such as TLR9, NLRP3, cGAS, P2X/P2Y receptors, GPR91 (DC), and FPR, which ultimately activate NLRP3 inflammasome leading to proinflammatory responses (Nakahira et al., 2015).

Ca2+ plays a significant role in driving mitochondrial physiology, dynamics, and signaling. A moderate increase in Ca2+ activates TCA cycle enzymes, whereas a significant increase causes the opening of mitochondrial permeability transition pores and leakage of solutes (<1.5 kDa), leading to different forms of cell death (Finkel et al., 2015). Higher Ca2+ levels also cause changes in mitochondrial dynamics, such as fission and rigidity. Ca2+ signaling plays a major role in the crosstalk between mitochondria and ER (Rowland and Voeltz, 2012).

Mitochondria contributes to signaling in cell death, inflammation and autophagy by offering themselves as a platform for various protein-protein interactions and as active participants by releasing mitochondrial factors (DAMPs). Mitochondria maintains a membrane potential, which, when altered, is perceived as a signal towards various cell fates, including inflammation, apoptosis, necrosis, and pyroptosis. The depolarized membrane releases mitochondrial DAMPs, each leading to various signaling outcomes ranging from inflammation to different forms of cell death, as outlined in Fig. 1. The extrinsic pathways mediated by death domain signaling lead to the translocation of anti-apoptotic factors and cleavage of pro-apoptotic factors by protein-protein interaction involving Bcl2, Bid, Bax, and AKAPs (A-kinase anchoring proteins) harbored in its outer membrane (Chandel, 2014).

Mitochondria are dynamic organelles that change their shape, size, and number in different physiological and pathological states by mitochondrial fission and fusion to maintain their shape and number. The mitochondrial dynamics is influenced by its fission, fusion, mitophagy, and transport which are highly conserved from yeast to human. Different cell types possess different structures and functions of mitochondria. The mitochondrial shape is determined by lipid and protein constitution that allows the influence of external signals and metabolic state in altering the shape of mitochondria ranging from a punctate to a tubular network. For instance, mitochondria are elongated in cultured cells, where the ATP requirement is high. Similarly, nutrient starvation and cellular stress result in fragmented mitochondria with decreased OXPHOS (Rambold and Pearce, 2018).

The initiation of mitochondrial fission occurs by the phosphorylation of cytosolic DRP-1 at S637 by environmental sensors such as FAK, ERK1/2, AKAP_1, and TBK-1, and subsequent recruitment to the mitochondrial outer membrane. However, phosphorylation by PKA causes its release from mitochondria and therefore inhibition of fission (Ul Fatima and Ananthanarayanan, 2023). Mitochondrial fission occurs at specific points along the mitochondrial surface determined by ER, wherein a GTPase-related protein, DRP1, assembles and winds around to pinch off the mitochondria with the aid of other proteins as adaptors, including MFF, Fis1, MID49–51, and Dynamin 2, resulting in fission. The relative levels of these proteins may be altered in a stressed cell. CluH is known to regulate the levels of MFF and MID49 mRNA (Yang et al., 2022). Growing evidence in mitochondrial research suggests that mitochondrial dynamics and cellular metabolism are coupled. For instance, mitochondria in cells predominantly undergoing OXPHOS are more condensed than in cells where glycolysis is predominant (Rossignol et al., 2004). Differential localization of fission points on mitochondria determines the fate of the fragmented mitochondria. If the fission occurs at the midpoint, it is likely to promote mitochondrial biogenesis, whereas the endzone fission results in its fragmentation (Kleele et al., 2021).

Mitochondrial fusion occurs in two steps; in the first step, the outer membrane fuses, followed by the inner membrane. The outer membrane fusion occurs with the help of MFN1 and 2 proteins in the outer membrane and tethers the two mitochondria to be fused. MFN2 is also present in ER. MFN1 and opa1, located in intermembrane space, bring about inner membrane fusion. Mitochondrial fusion is known to be regulated by mitochondrial Ca2+ levels and proteolytic cleavage of Opa1. Increased Ca2+ levels bind to Ca2+ sensing outer membrane proteins, Miro1/2, and inhibit MFN and therefore fusion. OPA1 exists in multiple isoforms. The long form, l-Opa-1, is proteolytically cleaved to a shorter form, s-OPA-1. The ratio of l-OPA-1 to s-OPA-1 regulates the fusion process. The exact mechanism of MFN1, MFN2, and OPA1 in the fusion process is unclear.

A balance of mitochondrial fission and fusion governs mitochondrial morphology, physiology, and, ultimately, mitochondrial immunometabolism. The mitochondrial dynamics are also regulated by nutrient availability. This observation appears to be an important link between metabolism and mitochondrial signaling, wherein mTOR and AMPK play an important role (Morita et al., 2017). mTOR complex senses nutrients and integrates extracellular and intracellular signals to promote anabolism and suppress catabolism. Active mTOR stimulates the translation of mitochondrial fission protein MTFP1, along with mitochondrially located DRP-1 resulting in mitochondrial fission (Morita et al., 2017). In the absence of glucose, cAMP is activated, further activating AMPK. The activation of AMPK results in increased mitochondrial biogenesis, which is mediated by the activation of PPAR-γ and PGC-1α (Herzig and Shaw, 2018). Therefore, nutrient starvation causes a hyperfusion of mitochondria, protecting against autophagy and cell death (Mishra and Chan, 2016).

Mitochondrial activity continuously generates ROS and necessitates robust redox homeostasis to counter oxidative stress. ROS generated in mitochondria is derived either as a by-product of respiration or as enzymatic activities of Xanthine/Xanthine oxidase, uncoupled nitric oxide synthases (NOS), cytochrome P-450 isoforms, α-ketoglutarate dehydrogenase (α-KGDH) complex and NADPH-dependent oxidases (NOXs). An imbalanced ROS level leads to the oxidation of biomolecules such as lipids, proteins, and low molecular weight thiols, contributing to oxidative stress and, eventually, mitochondrial dysfunction leading to cell death. The resulting superoxide radicals are reduced to form hydrogen peroxide, which is reduced by superoxide dismutases. Excess hydrogen peroxide could diffuse through membranes and contribute to redox signaling. The redox homeostasis is maintained by SOD (superoxide dismutase), catalase, NADPH, reduced glutathione, peroxiredoxins, and redox-active Cys. Please refer to (Handy and Loscalzo, 2012) for a detailed review.

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