A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis
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eLife Assessment
Verma and colleagues interrogate the mechanisms of phagosome maturation arrest during Mycobacterium tuberculosis infection. While cellular events that culminate in this arrest have been largely elucidated, involvement of other organelles, such as mitochondria, has not been highlighted mechanistically. In this valuable study, elements of mitochondrial quality control, such as mitophagy and mitochondrial-derived vesicles involvement, are shown to be paramount in the host-pathogen tussle. The evidence supporting the main conclusions is solid, based on multiple complementary approaches and appropriate controls, although some central mechanistic aspects of the proposed pathway remain only partially resolved.
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Abstract
Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes causes PMA. Here, we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) does not appear to impact mitochondrial quality. The p62 KD cells are restrictive to Mtb growth, triggered by an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and gets delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb ; the phenotype also replicated in p62/SQSTM1 knockout cells. Triggering MQC collapse in p62 KD cells further diminishes Mtb survival, signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.
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eLife Assessment
Verma and colleagues interrogate the mechanisms of phagosome maturation arrest during Mycobacterium tuberculosis infection. While cellular events that culminate in this arrest have been largely elucidated, involvement of other organelles, such as mitochondria, has not been highlighted mechanistically. In this valuable study, elements of mitochondrial quality control, such as mitophagy and mitochondrial-derived vesicles involvement, are shown to be paramount in the host-pathogen tussle. The evidence supporting the main conclusions is solid, based on multiple complementary approaches and appropriate controls, although some central mechanistic aspects of the proposed pathway remain only partially resolved.
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Reviewer #1 (Public review):
Summary:
This is an important and interesting manuscript that uncovers the cross-talk between mitochondrial quality control and phagosome maturation arrest imposed by Mtb.
A broader host pathogen (intracellular) question pertains to evading phagosomal maturation/arrest. While cellular events that culminate in this arrest have been largely elucidated, involvement of other organelles, such as mitochondria, has not been highlighted mechanistically. This manuscript paints a larger picture than the well-known conventional endolysosomal pathway and portrays a larger landscape involving elements of the mitochondrial quality control, such as mitophagy and mitochondrial-derived vesicles' involvement in the host-pathogen tussle.
Strengths:
The systematic characterisation to unravel the interplay between …
Reviewer #1 (Public review):
Summary:
This is an important and interesting manuscript that uncovers the cross-talk between mitochondrial quality control and phagosome maturation arrest imposed by Mtb.
A broader host pathogen (intracellular) question pertains to evading phagosomal maturation/arrest. While cellular events that culminate in this arrest have been largely elucidated, involvement of other organelles, such as mitochondria, has not been highlighted mechanistically. This manuscript paints a larger picture than the well-known conventional endolysosomal pathway and portrays a larger landscape involving elements of the mitochondrial quality control, such as mitophagy and mitochondrial-derived vesicles' involvement in the host-pathogen tussle.
Strengths:
The systematic characterisation to unravel the interplay between mitochondrial-related pathways and the endolysosomal system allows the authors to unearth some important findings.
Weaknesses:
The conclusions drawn require more robust experimentation and analysis.
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Reviewer #2 (Public review):
This manuscript examines the role of autophagy receptor proteins, particularly p62/SQSTM1, in regulating intracellular Mtb survival in human macrophages. Counterintuitively, depleting p62 reduces bacterial survival rather than enhancing it, pointing to a previously unrecognised mechanism. The authors demonstrate that in the absence of p62, mitochondrial quality is maintained through enhanced TOM20⁺ mitochondria-derived vesicle (MDV) biogenesis, dependent on MIRO1/MIRO2. During Mtb infection, these MDVs are redirected to bacterial phagosomes, promoting RAB7 recruitment, overcoming phagosome maturation arrest and facilitating lysosomal targeting of Mtb. In parallel, bacteria experience increased oxidative stress, further contributing to bacterial killing.
Strengths:
The mechanistic chain is built using …
Reviewer #2 (Public review):
This manuscript examines the role of autophagy receptor proteins, particularly p62/SQSTM1, in regulating intracellular Mtb survival in human macrophages. Counterintuitively, depleting p62 reduces bacterial survival rather than enhancing it, pointing to a previously unrecognised mechanism. The authors demonstrate that in the absence of p62, mitochondrial quality is maintained through enhanced TOM20⁺ mitochondria-derived vesicle (MDV) biogenesis, dependent on MIRO1/MIRO2. During Mtb infection, these MDVs are redirected to bacterial phagosomes, promoting RAB7 recruitment, overcoming phagosome maturation arrest and facilitating lysosomal targeting of Mtb. In parallel, bacteria experience increased oxidative stress, further contributing to bacterial killing.
Strengths:
The mechanistic chain is built using multiple complementary approaches, including genetic perturbation, redox biosensors, metabolic assays and microscopy. The use of primary human macrophages from multiple donors alongside established cell lines increases confidence that the phenotype is not cell-line specific. The replication clock experiment is particularly elegant and clearly demonstrates that the reduction in bacterial burden reflects enhanced killing rather than impaired bacterial replication. Overall, the study identifies an unexpected connection between mitochondrial quality control and phagosome maturation and provides a potentially important advance in our understanding of host-pathogen interactions.
Weaknesses:
The study remains entirely in vitro, and the phenotype is absent in mouse macrophages, limiting the immediate physiological and translational relevance of the findings. In addition, many of the central mechanistic conclusions rely heavily on colocalisation analyses, making it difficult to distinguish direct mechanistic relationships from associated trafficking events.
Overall, this is an interesting and technically strong study that uncovers a novel link between mitochondrial quality control and anti-mycobacterial defence. The mechanistic model is plausible and supported by substantial experimental work. However, several aspects of the proposed pathway require stronger experimental support before some of the broader conclusions can be fully justified.
Major points
(1) The central conclusion that TOM20⁺ MDVs are recruited to Mtb-containing phagosomes is based largely on microscopy and colocalisation analyses. Additional orthogonal approaches would strengthen this key aspect of the study and help establish the nature of the vesicles recruited to bacterial phagosomes.
(2) The proposed mechanism whereby TOM20⁺ MDVs facilitate RAB7 recruitment and reverse phagosome maturation arrest remains incompletely demonstrated. While the MIRO1/2 and RAB7 knockdown experiments support the model, they do not directly establish a causal link between MDV recruitment and phagosomal RAB7 acquisition. Additional experiments addressing this step would considerably strengthen the manuscript.
(3) The absence of a phenotype in mouse macrophages raises important questions regarding the conservation and physiological relevance of the proposed mechanism. The authors should discuss possible explanations for this species-specific effect and, if feasible, provide additional experimental insight into the basis of this difference.
(4) The conclusion that mitochondrial quality is maintained despite impaired p62-dependent mitochondrial turnover is based primarily on mitochondrial content, membrane potential, ROS measurements and Seahorse analysis. These are informative but relatively indirect measurements. Additional assessment of mitochondrial turnover by mitophagy would strengthen this aspect of the study.
(5) The proteins studied throughout the manuscript (p62/SQSTM1, NDP52, OPTN, TAX1BP1 and NBR1) are generally classified as selective autophagy receptors rather than adaptors. The terminology should be corrected throughout the manuscript.
Minor points:
(1) Several conclusions throughout the manuscript are based primarily on colocalisation analyses. The limitations of these approaches should be acknowledged explicitly.
(2) The discussion would benefit from a clearer consideration of how the proposed mechanism relates to established pathways regulating phagosome maturation arrest during Mtb infection.
(3) The authors may wish to comment on whether enhanced MDV biogenesis could represent a broader host defence mechanism against intracellular pathogens beyond Mtb.
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