NCoR1: a key player regulating mycobacterium tuberculosis pathogenesis

ABSTRACT

Mycobacterium tuberculosis (Mtb) employs a multifaceted arsenal to elude host defense mechanisms, including those associated with autophagy and lysosome function. Within the realm of host-pathogen interactions, NCOR1, a well-recognized transcriptional co-repressor, is known to associate with a multitude of protein complexes to effect the repression of a diverse spectrum of genes. However, its role in regulating macroautophagy/autophagy, lysosome biogenesis, and, by extension, Mtb pathogenesis remains unexplored. The depletion of NCOR1 assumes a pivotal role in the control of the AMPK-MTOR-TFEB signaling axis, thereby fine-tuning cellular ATP homeostasis. This finely orchestrated adjustment further alters the profile of proteins involved in autophagy and lysosomal biogenesis through its master regulator, TFEB, culminating in the increased Mtb survival within the host milieu. Furthermore, the treatment of NCOR1-depleted cells with either rapamycin, antimycin A, or metformin demonstrates a capacity to restore the TFEB activity and LC3-II levels, consequently restoring the capacity of host cells to clear Mtb. Additionally, exogenous NCOR1 expression rescues the AMPK-MTOR-TFEB signaling axis and essentially the autophagic induction machinery. Overall, these findings demonstrate a crucial role of NCOR1 in regulating Mtb pathogenesis within myeloid cells and sheds light toward its involvement in the development of novel host-directed therapies.

Main

The dynamics of NCOR1 expression during active Mtb infection in human PBMCs and its restoration upon extended anti-mycobacterial therapy provide useful insights into the role of NCOR1 in Mtb pathogenesis. NCOR1 deficiency impedes the clearance of pathogenic as well as nonpathogenic strains of mycobacterium, i.e., H37Rv and M. smegmatis, respectively in THP-1 derived human macrophages and the cDC1 dendritic cell (DC1940) line in vitro, in bone marrow-derived macrophages/BMDMs ex vivo and in vivo in myeloid cell-specific ncor1 KO mice (ncor1^Myeloid-/-) [1]. The mechanistic underpinnings of NCOR1-mediated control over Mtb growth in vivo depicted increased inflammation, culminating into elevated disease burden. Whereas, in vitro and ex vivo studies considerably show its dependence on the regulation of cellular energy homeostasis pathways, in particular the AMPK-MTOR-TFEB (transcription factor EB) signaling axis (Figure 1). Comprehensive transcriptome analysis of Mtb-infected NCOR1-depleted human macrophages reveals downregulation of key genes associated with the autophagy pathway, including ATG3, ATG5, and ATG13. Moreover, a significant reduction in the LC3-II:LC3-I ratio is observed in the NCOR1-depleted condition, suggesting a compromised autophagic flux. To corroborate the same, bafilomycin A₁ treatment was done; this aggravates the NCOR1-mediated support of intracellular Mtb growth, confirming its role in autophagy induction. Furthermore, our data revealed a pronounced downregulation of critical proteins associated with autophagosomal formation and nucleation, specifically, the ATG12–ATG5 conjugate and BECN1. These findings underscore the substantial impact of NCOR1 KD on the regulation of key molecular players within the autophagy machinery. Mechanistically, NCOR1 regulates TFEB levels, with its absence hampering autolysosome formation, heightening susceptibility to Mtb pathogenesis. To establish a relationship between them, the expression kinetics of TFEB were attained. Interestingly, both NCOR1 and TFEB exhibit similar expression patterns in response to Mtb infection, suggesting a plausible interconnection between these two entities. Considering TFEB’s direct role in lysosomal biogenesis, LAMP1 expression levels were also determined. LAMP1 protein expression is severely compromised in NCOR1-ablated conditions and provides enough evidence for defunct lysosomal biogenesis occurring under this situation.

Figure 1.

Schematic illustration depicting Mtb survival in NCOR1-depleted myeloid cells as compared to controls. NCOR1 ablation promotes heightened OXPHOS and as a result more ATP. The higher cellular ATP levels inactivate AMPK and induce MTOR. The active MTOR inhibits translocation of TFEB into the nucleus and thus inhibits the expression of autophagy induction and lysosomal biogenesis genes. These events in synchronization promote more Mtb survival in NCOR1-depleted myeloid cells.

NCOR1’s influence on cellular energy processes, including ATP synthesis, are also found to be significant. A surge in ATP during Mtb infection leads to changes in host cell oxidative phosphorylation (both basal and maximal respiration), which Mtb exploits for survival. AMPK, a key sensor for dynamic ATP concentration in cells, responds inversely to the cellular energy state with respect to MTOR, a well-known regulator of autophagy, negatively regulated by phosphorylated AMPK. NCOR1 is found to be a positive regulator of the AMPK-MTOR-TFEB signaling cascade, inducing autophagy and promoting host defense mechanisms. Upon NCOR1 depletion, AMPK becomes inactive, leading to MTOR induction and subsequent inhibition of TFEB translocation to the nucleus. TFEB autonomously orchestrates the autophagy machinery and lysosomal biogenesis, endowing Mtb with survival advantages in NCOR1-depleted conditions (Figure 1). To further confirm the role of the NCOR1-mediated increase in ATP levels, leading to heightened Mtb infection, antimycin A, an inhibitor of complex III of the electron transport chain/ETC was utilized. ATP depletion results in reduced bacterial burden in NCOR1-ablated conditions with recovered AMPK activity and decreased levels of p-MTOR, leading to TFEB restoration. Similar rescue experiments were also conducted with the AMPK inhibitor metformin. In addition, to show that NCOR1 is a key candidate to control the pathogenesis of Mtb in the host and can be a putative target in controlling Mtb infection, exogenous NCOR1 was overexpressed in NCOR1-depleted macrophages. The exogenous delivery of NCOR1 proves sufficiently efficacious in restoring the compromised AMPK-MTOR signaling axis, leading to the recovery of LC3-II levels. Moreover, exogenous NCOR1 complementation rescues the NCOR1 KD macrophages from the susceptibility to Mtb infection. Overall, NCOR1’s dual roles are proposed: interaction with TFEB for functionality or stability, and regulation of AMPK-MTOR signaling. In sum, the study reveals NCOR1’s direct involvement in Mtb survival through optimal autolysosomal regulation.

While this study provides valuable insights, there are some limitations. The precise mechanisms by which NCOR1 regulates AMPK, MTOR, and TFEB need further elucidation. Additionally, the study primarily focused on myeloid cells, and the role of NCOR1 in other cell types involved in TB immunity should be explored. Finally, clinical studies are needed to validate the relevance of these findings in human TB cases.

Funding Statement

This work was supported by grants from SERB-India (grants EMR/2016/000717) and also from Department of BioTechnology-India (grants BT/PR15908/MED/12/725/2016). ILS provided intramural core grants and infrastructure to carry out the study.

Disclosure statement

No potential conflict of interest was reported by the author(s).