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Mitochondrial function and innate immunity are intimately linked; however, the mechanisms how mitochondrion-shaping proteins regulate innate host defense remains largely unknown. Herein we show that mitofusin-2 (MFN2), a mitochondrial fusion protein, promotes innate host defense through the maintenance of aerobic glycolysis and xenophagy via hypoxia-inducible factor (HIF)-1α during intracellular bacterial infection. Myeloid-specific MFN2 deficiency in mice impaired the antimicrobial and inflammatory responses against mycobacterial and listerial infection. Mechanistically, MFN2 was required for the enhancement of inflammatory signaling through optimal induction of aerobic glycolysis via HIF-1α, which is activated by mitochondrial respiratory chain complex I and reactive oxygen species, in macrophages. MFN2 did not impact mitophagy during infection; however, it promoted xenophagy activation through HIF-1α. In addition, MFN2 interacted with the late endosomal protein Rab7, to facilitate xenophagy during mycobacterial infection. Our findings reveal the mechanistic regulations by which MFN2 tailors the innate host defense through coordinated control of immunometabolism and xenophagy via HIF-1α during bacterial infection.

Autophagy is known to be a vital homeostatic defense process that controls mycobacterial infection. However, the relationship between autophagy response and the virulence of Mycobacterium abscessus strain UC22 has not been reported. Here, we demonstrate that M. abscessus induces autophagy and inhibits autophagy flux in murine macrophages. Further, the rough variant of M. abscessus, UC22 that is a highly virulent clinical isolate, significantly inhibited autophagic flux than the smooth variant of M. abscessus ATCC 19977. In addition, it was noticed that the intracellular survival of UC22 is significantly enhanced by blocking the autophagosome-lysosome fusion in macrophages compared to the smooth variant. However, Mycobacterium smegmatis did not block autophagy flux in murine macrophages. Besides, we confirmed that the lipid components of M. abscessus UC22 play a role in autophagosome formation. These data suggest that the virulent M. abscessus might be able to survive and grow within autophagosomes by preventing the autophagosome-lysosome fusion and their clearance from the cells., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mycobacterium abscessus (MAB) is a species of nontuberculous mycobacteria (NTM) and a major causative pathogen of pulmonary diseases especially in patients with cystic fibrosis. MAB infection is notoriously difficult to treat because of its intrinsic or inducible resistance to most antibiotics. The rough (R) morphotype of MAB, lacking cell surface glycopeptidolipids (GPLs), is associated with more severe and persistent infection than the smooth (S) type; however, the mechanisms underlying the R type's virulence and the relation with GPLs remain unclear. In this study, we found that R-type MAB is much more proapoptotic than the S type, as a result of GPL-mediated inhibition of macrophage apoptosis. Polar GPLs inhibited an apoptotic response (induced by proapoptotic stimuli) by suppressing ROS production and the cytochrome c release and by preserving mitochondrial transmembrane potential. Furthermore, GPLs were found to be targeted to mitochondria and interacted with cyclophilin D; their acetylation was essential for this interaction. Finally, GPLs inhibited the intracellular growth and bacterial spreading of R-type MAB among macrophages via apoptosis inhibition. These findings suggest that GPLs limit MAB virulence by inhibiting apoptosis and the spread of bacteria and therefore provide a novel insight into the mechanism underlying virulence of MAB.

Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨ m ) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨ m collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

Mycobacteria encounter many different cells during infection within their hosts. Although alveolar epithelial cells play an essential role in host defense as the first cells to be challenged upon contact with mycobacteria, they may contribute to the acquisition of mycobacterial virulence by increasing the expression of virulence or adaptation factors prior to being ingested by macrophages on the side of pathogens. From this aspect, the enhanced virulence of nonpathogenic Mycobacterium smegmatis (MSM) passed through human alveolar A549 epithelial cells (A-MSM) was compared to the direct infection of MSM (D-MSM) in THP-1 macrophages and mouse models. The intracellular growth rate and cytotoxicity of A-MSM were significantly increased in THP-1 macrophages. In addition, compared to D-MSM, A-MSM induced relatively greater interleukin (IL)-1β, IL-6, IL-8, IL-12, TNF-α, MIP-1α, and MCP-1 in THP-1 macrophages. As a next step, a more persistent A-MSM infection was observed in a murine infection model with the development of granulomatous inflammation. Finally, 58 genes induced specifically in A-MSM were partially identified by differential expression using a customized amplification library. These gene expressions were simultaneously maintained in THP-1 infection but no changes were observed in D-MSM. Bioinformatic analysis revealed that these genes are involved mainly in bacterial metabolism including energy production and conversion, carbohydrate, amino acid, and lipid transport, and metabolisms. Conclusively, alveolar epithelial cells promoted the conversion of MSM to the virulent phenotype prior to encountering macrophages by activating the genes required for intracellular survival and presenting its pathogenicity.