Mycobacterium tuberculosis induces host autophagic ferritin degradation for enhanced iron bioavailability and bacterial growth

ABSTRACT

To prevent Mycobacterium tuberculosis (Mtb) infection, the host can restrict iron availability via iron-chelation proteins such as ferritin, which is a key regulator of iron homeostasis in macrophages. However, how Mtb escapes the nutrient immunity mediated by ferritin and survives in the infected host despite ongoing immune responses remains unclear. Our recent findings demonstrated that Mtb exploited the autophagic degradation of ferritin mediated by a selective autophagy receptor, nuclear receptor coactivator 4 (NCOA4), in macrophages for enhanced iron bioavailability and bacterial growth. Further, we found that ferritin degradation in macrophages is involved in human tuberculosis disease progression. Mechanistically, Mtb-induced ferritin degradation is initiated by p38 protein kinase (p38) and AKT serine/threonine kinase 1 (AKT1) signaling and mediated by a tripartite motif containing 21 (TRIM21)-dependent proteasomal degradation cascade. Finally, we used a mouse model with NCOA4-deficient myeloid cells to confirm their role in resistance to Mtb infection. Our study thus identifies modulation of host ferritin metabolism as a novel Mtb strategy for intracellular growth, which may provide a potential target for host-directed therapy against tuberculosis.

Abbreviations

Mtb, Mycobacterium tuberculosis; TB, tuberculosis; NCOA4, nuclear receptor coactivator 4; p38, p38 protein kinase; AKT1, AKT serine/threonine kinase 1; TRIM21, tripartite motif containing 21; FTH1, ferritin heavy chain 1; FTL, ferritin light chain; HERC2, HECT and RLD domain containing E3 ubiquitin protein ligase 2.

Iron is an essential nutrient element for both hosts and pathogens, including Mycobacterium tuberculosis (Mtb). During infection, the host utilizes chelation of iron in ferritin and lactoferrin as a crucial defense strategy to prevent intracellular pathogens from accessing iron. Consistent with this notion, ferritin deficiency in myeloid cells or bone marrow increases the susceptibility of Mtb infection. In contrast, ferritin degradation could benefit pathogens by supplying bioavailable iron. Some pathogens, such as Escherichia coli and Ehrlichia chaffeensis, utilize ferritinophagy, a selective type of autophagy mediating lysosomal degradation of ferritin, to access host ferritin-stored iron for their intracellular survival and growth. Understanding whether and how Mtb evades the nutrient immunity mediated by ferritin is important for enabling the development of new therapies for tuberculosis (TB). In our recently published study, we uncovered that Mtb hijacks host (tripartite motif containing 21) TRIM21- and nuclear receptor coactivator 4 (NCOA4)-dependent ferritinophagy to enhance intracellular growth (Figure 1) [1].

Figure 1.

Mycobacterium tuberculosis hijack host ferritinophagy to enhance intracellular growth. Mtb infection promotes the transcriptional expression of TRIM21, which functions as an E3 ligase for the proteasomal degradation of HERC2 in macrophages. HERC2-mediated proteolysis inhibits the turnover of NCOA4 protein, which leads to an enhancement of NCOA4-mediated ferritin degradation and supplies free iron to the intracellular bacteria. Consistently, NCOA4 deficiency in myeloid cells increases host resistance to Mtb infection.

Host ferritin in macrophages plays a dual role in Mtb infection

We first found increased levels of ferritin in peripheral blood mononuclear cells (PBMCs) isolated from patients with active TB, as compared to the healthy controls and non-TB pneumonia patients. Macrophages unregulated ferritin expression upon Mtb infection. By using a dual-color reporter Mtb whose live or dead status within macrophages can be differentiated by fluorescence-activated cell sorting, we found ferritin heavy chain 1 (FTH1) and ferritin light chain (FTL) were significantly increased in macrophages harboring live Mtb compared with uninfected macrophages or those harboring dead Mtb. In agreement with others, we observed that FTH1 knockdown increased intracellular Mtb growth, which could be abrogated by iron chelation using deferoxamine or iron supplementation with ferrous lactate. In contrast, preloaded ferritin in macrophages supplied iron for intracellular Mtb growth when free iron was withdrawn from macrophages. Thus, we redefined a dual role for host ferritin in Mtb infection depending on the availability of iron.

NCOA4-mediated ferritin degradation in macrophages is essential for Mtb infection

Next, we investigated whether and how Mtb hijacks ferritin metabolism for its intracellular growth. Ferritin degrades and releases bound iron through a NCOA4-mediated ferritinophagy. We observed that NCOA4 was upregulated and directly interacted with FTH1 in Mtb-infected macrophages, while NCOA4 knockdown or deficiency resulted in ferritin accumulation, decreased intracellular free iron, and Mtb growth. The decreased iron acquisition by Mtb isolated from NCOA4 knockdown or NCOA4-deficient macrophages was validated by measuring the free iron and expression of iron-response genes in these bacteria. Furthermore, we compared NCOA4 and FTH1 expression in the pulmonary tissues from patients undergoing therapeutic resection for advanced TB and diagnostic biopsy specimens for earlier-stage TB using quantitative immunohistochemistry. We observed a higher percentage of NCOA4-positive but a lower percentage of FTH1-positive macrophages within granulomas in the therapeutic resection tissues than in the diagnostic biopsy specimens. Most importantly, in accordance with the in vitro findings, our in vivo results confirmed that NCOA4-deficiency in myeloid cells contributes to host resistance against Mtb infection. Specifically, following aerosol infection with Mtb, the bacterial loads and immune-cell infiltrations in the lungs of Lyz2^creNcoa4^fl/fl mice were significantly reduced compared with those in Ncoa4^fl/fl control mice. Thus, our in vitro and in vivo results indicate that NCOA4-mediated ferritinophagy is beneficial for intracellular Mtb growth by increasing iron availability.

Ferritinophagy is initiated by p38/AKT1-signaling and mediated by a novel TRIM21-depdendent proteasomal degradation cascade

The finding that induction of ferritinophagy via NCOA4 is regulated by HECT and RLD domain containing E3 ubiquitin protein ligase 2 (HERC2)-mediated proteolysis during erythropoiesis prompted us to investigate whether Mtb infection inhibits HERC2 expression in macrophages. As expected, we found that Mtb infection reduced the level of cytoplasm HERC2, which inhibited the turnover of NCOA4 in macrophages. HERC2 functions as an E3 ligase to mediate proteolysis, but the regulation of HERC2 expression has not been elucidated. By using the pan-inhibitor of protein synthesis (cycloheximide), the proteasome inhibitor (MG-132), and the lysosomal inhibitors (chloroquine and bafilomycin A1), we concluded that the turnover of HERC2 depends on proteasomal degradation. Further, we employed co-immunoprecipitation coupled with mass spectrometry to screen the E3 ligase involved in the proteasomal degradation of HERC2 in macrophages during Mtb infection. Through immunoprecipitation, confocal microscopy, small interfering RNA (siRNA), and signaling pathway inhibitors, we identified TRIM21 as the E3 ligase modifying HERC2, whose transcriptional expression is induced by Mtb infection mainly through p38 and AKT1 signaling pathways.

In summary, our in vitro and in vivo findings demonstrated that Mtb hijacks host NCOA4-mediated ferritinophagy in macrophages to promote its intracellular growth via increased iron bioavailability. This novel evasion mechanism is initiated by p38 and AKT1 signaling pathways and involves a TRIM21-dependent proteasomal degradation.

Funding Statement

The work was supported by the Science and Technology Project of Guangdong Province [grants 2023A1515010351]; Natural Science Foundation of China [82100015]; Shenzhen Bay Laboratory Open Project [grant SZBL2020090501010]; Guangdong Provincial Key Laboratory of Regional Immunity and Diseases [grant 2019B030301009]; Natural Science Foundation of China [grants 82130066, 91942315, 2022YFC2302900].

Disclosure statement

No potential conflict of interest was reported by the authors.