TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages, 2022, Schuster et al

[darrellpf]

"Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB–Irg1–itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32–BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB’s role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo."

https://www.nature.com/articles/s42255-022-00605-w

 

[Hutan]

Interesting paper, confirming the itaconate shunt as a means of controlling bacteria in macrophages and demonstrating that measuring levels of itaconate in cells is straight-forward.

When we followed the fate of glucose-derived carbon downstream of citrate, we found that TFEB activation routed carbon flux primarily into itaconate, an integral metabolite of the pro-inflammatory macrophage response23, but not palmitate (Fig. 1h). With this itaconate-fuelling response, TFEB promotes a TCA-cycle state that is normally engaged in macrophages upon bacterial stimulation (Extended Data Fig. 1h)22. Thus, our data reveal a previously unappreciated link between TFEB and a biosynthetic TCA-cycle state in macrophages.

TFEB’s altered carbon funnelling resulted in substantially elevated cellular itaconate levels (Fig. 2a), as measured by liquid chromatography coupled to mass spectrometry (LC–MS). Similar to the genetic model, the activation of endogenous TFEB by the TFEB activator (TFEBa) 2-hydroxypropyl-β-cyclodextrin24 enhanced glucose-derived carbon fuelling of itaconate and increased itaconate levels (Fig. 2b and Extended Data Fig. 2a,b). Itaconate production was also induced by TFEB activation after lysosomal inhibition via the V-ATPase inhibitor bafilomycin A1 (Baf)25 (Fig. 2c and Extended Data Fig. 2a). Thus, TFEB activation alone is sufficient to produce itaconate without additional need for a pro-inflammatory macrophage signal.

 

[Hutan]

Itaconate produced by mitochondria can be moved into vacuoles containing pathogen bacteria where it inhibits pathogen proliferation.

Itaconate can be transferred from mitochondria into the pathogen-containing vacuole via Irg1-Rab32–BLOC3-driven organelle interactions and directly inhibit Salmonella growth9,23 (Extended Data Fig. 4a). To assess whether vacuolar itaconate mediates the growth suppressive TFEB-effect, we infected macrophages with Salmonella carrying previously described sensor plasmids (GFP-ITA or NanoLuc-ITA) that allow the visualization and measurement of vacuolar itaconate9 (Fig. 4h and Extended Data Fig. 5a). Although, Salmonella Typhimurium can partially counteract vacuolar itaconate transport by cleaving Rab32 (ref. 9) (Extended Data Fig. 5b), we found that TFEB activation increased the percentage of itaconate-exposed pathogens (Fig. 4h) and raised vacuolar itaconate levels by 2.6-fold in comparison to control cells

In primary macrophages, our results identify the TFEB-driven Irg1–itaconate axis as a lysosome-to-mitochondria communication pathway that controls a cell-autonomous antibacterial defence mechanism to protect the phago-lysosomal compartment from being exploited as bacterial proliferation niche. Our data indicate that the TFEB–Irg1–itaconate pathway exerts its antimicrobial function primarily in the vacuole: functions of cytosolic itaconate45,46, TFEB or Hps4, and potential functional interactions between those systems47, may play additional roles or modulate the vacuolar response

How itaconate controls Salmonella proliferation mechanistically is currently unclear, but probably relates to its ability to inhibit selected metabolic enzymes. This includes key enzymes in the glyoxylate shunt and propionate metabolism that different pathogens rely on for intra-macrophage growth during acute and chronic infections23,35,49,50,51.

Most of the authors of this article are based in Freiburg, Germany.

Additional activation of TFEB by LPS/IFNγ treatment or LPS signalling alone further augmented itaconate levels (Fig. 2d and Extended Data Fig. 2d), without affecting iNOS expression, a negative regulator of itaconate synthesis in activated BMDMs

I thought the mention of iNOS as an inhibitor of itaconate synthesis was interesting, given its possible link to brain and CNS pathologies.

 

[Hutan]

Here's an explanation of how TFEB becomes activated (TFEBa) so it can then potentially go on to turn on the itaconate shunt):
(Basically, the TFEB molecule sits in the cytosol, inactive. Then, when there is some adverse condition e.g. starvation , infection, ROS, ER stress or mitochondrial damage, it is dephosphoyrlated and moves to the nucleus, becoming active. mTOR, ERK2 and PKC are involved in TFEB becoming active.

Emerging role of transcription factor EB in mitochondrial quality control 2020

TFEB activity and subcellular localization is strictly controlled by post-translational modifcations, protein-protein interactions and spatial organization. Under resting condition, TFEB is retained in the cytoplasm in an inactive, phosphorylated state [25]. However, under adverse conditon, like starvation, lysosomal storage, infection, mitochondrial damage or ER stress, TFEB moves to the nucleus and activates after dephosphorylation [26]. It has been confirmed that the activity and subcellular localization of TFEB in cells are determined by its phosphorylation status. Furthermore, the phosphorylation status of TFEB is mainly determined by the 142th (Ser142) and 211th (Ser211) sites of its Serine residues. When Ser142 and Ser211 are phosphorylated at the same time, TFEB is in an inactive state. When either or both of Ser142 and Ser211 are dephosphorylated, TFEB is transcriptionally activated and transported to nucleus to exert its regulatory function [[27], [28], [29]]. In addition, studies have shown that the main protein kinasesinvolved in TFEB phosphorylation modification are the serine/threonine kinase mammalian target of rapamycin (mTOR) [27], extracellular regulated protein kinases 2 (ERK2) [30] and protein kinase C (PKC) [31].

So, there are potentially quite a number of problems that could result in TFEB becoming active, although I'm not sure if they all result in TFEB turning on the itaconate shunt. (The paper that is the subject of this thread reported their finding that
" TFEB activation alone is sufficient to produce itaconate without additional need for a pro-inflammatory macrophage signal.")