Tuberculosis: autophagy is a part of the answer

Mycobacterium tuberculosis (Mtb) caused 1.6 million deaths in 2022. According to WHO, this is an increase despite underreporting of new tuberculosis (TB) cases due to COVID19. Studies with Mtb are notoriously laborious due to slow replication of the bacilli and long disease course when appropriately modeled in animals. In 2004 autophagy was reported as a promising new cell-autonomous defense against Mtb.¹ This received initial validation in mouse models in 2012,^2,3 but was challenged in 2015,⁴ which snuffed the initial excitement. Now, a careful 2023 study in this issue⁵ demonstrates that the conclusions dismissing autophagy were premature and that canonical autophagy or, alternatively, related noncanonical processes, confer protection against TB. This should rekindle interest in the therapeutic potential of autophagy for TB nearly twenty years since the initial report.¹

Canonical autophagy is a homeostatic process that impacts human health; it cleanses cytoplasm, turns over macromolecules and organelles, and eliminates invading microbes.⁶ Autophagy has effects on immunity and in principle suppresses inflammation, which causes pathology when excessive.⁶ One of the early reports indicating that autophagy is an antimicrobial process was a study showing that induction of autophagy restricts virulent Mtb in murine and human macrophages, the cell type where this pathogen normally replicates.¹ Subsequent in vivo studies reported high mortality and excessive inflammation in murine models of TB^2,3 employing conditional inactivation of Atg5 (Atg5^fl/flLysM-Cre) in myeloid cells. Atg5 is one of the core autophagy genes (Figure). These studies used low dose aerosol exposure causing chronic disease, modeling typical presentation of TB.^2,3 A subsequent analysis⁴ used conditional knockouts in multiple autophagy genes. That study confirmed that Atg5^fl/flLysM-Cre mice were particularly susceptible to Mtb and established that excessive neutrophilic inflammation was responsible for pathology and mortality.⁴ However, In the same study, mice with conditional knockouts in a panel of other autophagy genes, Atg14, Ulk1/2 (Atg1), and the enzymatic components Atg3, Atg7, Atg12, and Atg16L1 carrying out LC3-lipidation (also referred to as membrane atg8ylation;⁶ Figure) did not succumb to Mtb infection within 80 days post-infection.⁴ This report had a chilling effect on the prospects that autophagy may be of use in controlling Mtb, as reflected in its accompanying News and Views article entitled “Tuberculosis: autophagy is not the answer”.⁷

Figure:

Canonical and non-canonical ATG-dependent processes contribute to control of M. tuberculosis.

In a new study, Golovkine et al.,⁵ considered the possibility that conditional inactivation of floxed Atg genes with the LysM-Cre driver might have been incomplete for certain genes. They painstakingly titrated Cre-driven LoxP-excision events, by increasing Cre recombinase gene dosage in mice bread to be homozygous for LysM-Cre (LysM-Cre^+/+) instead of carrying only one LysM-Cre allele (LysM-Cre^+/−) customarily considered to be sufficient for Cre-driven Lox-excision. This has created a more complete inactivation of Atg7 and Atg16L1 genes, resulting in increased susceptibility of LysM-Cre^+/+Atg7^fl/fl and LysM-Cre^+/+Atg16L1^fl/fl mice to Mtb compared to their Cre⁻ littermates. In parallel, LysM-Cre^+/−Atg7^fl/fl showed no increased susceptibility to infection but LysM-Cre^+/−Atg16L1^fl/fl mice carrying only one LysM-Cre allele displayed increased susceptibility to Mtb, missed in the previous study⁴ due to shorter duration of observations. Additional experiments by Golovkine et al.,⁵ confirmed in LysM-Cre^+/+Atg7^fl/fl and LysM-Cre^+/+Atg16L1^fl/fl mice all prior observations regarding increased lesions and neutrophilic infiltration reported for Atg5^fl/flLysM-Cre mice ^2–4 and observed dynamic changes in bacterial burden. Thus, these careful studies demonstrate that multiple autophagy genes are needed for disease control in the mouse model of Mtb infection.

The study by Golovkine et al.,⁵ also reaffirmed the unique phenotype of Atg5 relative to other atg8ylation genes, whereby Mtb-infected mice depleted for Atg5 in myeloid cells died faster than the properly depleted Atg7 or Atg16L1 mice. Meanwhile, the unique role of Atg5 in vivo has been replicated in a recent study by Wang et al.,⁸. Mechanistically, Atg5 has moonlighting jobs beside its function in canonical autophagy. It helps maintain functional lysosomes and affects exocytosis.⁸ In the absence of Atg5, instead of the canonical Atg12-Atg5 conjugate (Figure), Atg12 engages in the formation of the noncanonical Atg12-Atg3 conjugate which binds ALIX thus redirecting this ESCRT protein from lysosomal repair tasks to exocytic events.⁸ In neutrophils, this causes excessive activation and degranulation,⁸ explaining in part the unique standing of Atg5 relative to other components of the atg8ylation machinery.

Taken together, the above studies^5,8 reaffirmed the interest in autophagy but also left the door open for contributions of noncanonical processes. Importantly, the revamped analysis with better excision of floxed alleles was limited only to Atg7 and Atg16L1.⁵ These Atgs are specific for the atg8ylation process⁶ conjugating mammalian Atg8s to membrane phospholipid phosphatidylethanolamine, commonly referred to as LC3-lipidation. Atg8ylation is not restricted to canonical autophagy and participates in a variety of membrane stress and remodeling responses, the extent of which is only beginning to be unraveled, encompassing LC3-associated phagocytosis (LAP) and a variety of other processes.⁶

LAP has been previously implicated in control of Mtb using CpsA mutant bacilli but only during innate responses, as IFN-γ and adaptive immunity abrogated CpsA effects both ex and in vivo.⁹ IFN-γ induces autophagy.¹ A new study in this issue by Aylan et al.,¹⁰ aimed to assess relative contributions of LAP vs canonical autophagy in control of Mtb by comparing Mtb Esx and CpsA mutants. ESX-1 Type 7 secretion system is important for Mtb escape from the phagosome⁴ whereas CpsA inhibits NOX2⁹ needed for LAP activation. Somewhat complicating the task, CpsA binds canonical autophagy cargo receptors NDP52 and TAX1BP1.⁹ It is also not clear how CpsA enters the host cytosol and this might depend on permeabilization by Esx. Aylan et al.,¹⁰ compared human iPSC macrophages lacking ATG14, considered to be specific for canonical autophagy, and ATG7 affecting both autophagy and LAP. The results showed some predictable and some unpredictable outcomes. As one might anticipate based on the multifaceted outputs of atg8ylation (Figure), the loss of ATG7 permitted growth of all strains tested (wild type Mtb and its CpsA and Esx mutants) thus not discerning between canonical autophagy and non-canonical effects of atg8ylation. Further, Golovkine et al.,⁵ observed that murine macrophages defective in atg8ylation, i.e. mutants in Atg5, Atg7 and Atg16L1, could not limit Esx-mediated phagosome disruption and cytosolic access by mycobacteria. One consequence of this was that cell death of atg8ylation-deficient macrophages infected with Mtb switched from less-inflammatory apoptosis to a more pro-inflammatory necrotic type of cell death, reminiscent of previously reported observations.³ This was corroborated by Aylan et al.,¹⁰ in human iPSC-derived macrophages defective in ATG7. Further surprises come from the observations by Aylan et al., that a loss of ATG14 (considered to be a canonical autophagy gene) restricted the Esx mutant, not anticipated to be subject to canonical autophagy since it would be confined to the phagosome.¹⁰ This led investigators to propose a further branching of processes controlled by ATGs, whereby ATG14 affects phagosomal maturation.¹⁰

The field of autophagy has evolved ⁶ since the initial report on its effects on Mtb.¹ Canonical autophagy is a part of a broader membrane stress and remodeling response (including LAP) controlled by the atg8ylation machinery.⁶ The study by Golovkine et al.,⁵ highlights the in vivo role of atg8ylation components contributing to canonical autophagy. The studies by Aylan et al.,¹⁰ and Wang et al.,⁸ highlight both canonical and noncanonical processes. At the moment the journey to autophagy-based interventions in TB may appear more complicated. However, the road is wide open. Directions may need to be refined and pharmacological targets redefined as our knowledge of the ATG system grows and our understanding of how it influences host-pathogen interactions matures.