Galectin-8 senses phagosomal damage and recruits selective autophagy adapter TAX1BP1 to control Mycobacterium tuberculosis infection in macrophages

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:italic>Mycobacterium tuberculosis</jats:italic> (Mtb) infects a quarter of the world and causes the deadliest infectious disease worldwide. Upon infection, Mtb is phagocytosed by macrophages and uses its virulence-associated ESX-1 secretion system to modulate the host cell and establish a replicative niche. We have previously shown the ESX-1 secretion system permeabilizes the Mtb-containing phagosome and that a population (~30%) of intracellular Mtb are recognized within the cytosol, tagged with ubiquitin, and targeted to the selective autophagy pathway. Despite the importance of selective autophagy in controlling infection, the mechanisms through which macrophages sense and respond to damaged Mtb-containing phagosomes remains unclear. Here, we demonstrate that several cytosolic glycan-binding proteins, known as galectins, recognize Mtb-containing phagosomes. We found that galectins-3, -8, and -9 are all recruited to the same Mtb population that colocalizes with selective autophagy markers like ubiquitin, p62, and LC3, which indicates Mtb damages its phagosomal membrane such that cytosolic host sensors can recognize danger signals in the lumen. To determine which galectins are required for controlling Mtb replication in macrophages, we generated CRISPR/Cas9 knockout macrophages lacking individual or multiple galectins and found that galectin-8<jats:sup>-/-</jats:sup> and galectin-3/8/9<jats:sup>-/-</jats:sup> knockout macrophages were similarly defective in targeting Mtb to selective autophagy and controlling replication, suggesting galectin-8 plays a privileged role in anti-Mtb autophagy. In investigating this specificity, we identified a novel and specific interaction between galectin-8 and TAX1BP1, one of several autophagy adaptors that bridges cargo and LC3 during the course of autophagosome formation, and this galectin-8/TAX1BP1 interaction was necessary to efficiently target Mtb to selective autophagy. Remarkably, overexpressing individual galectins increased targeting of Mtb to antibacterial autophagy and limited Mtb replication. Taken together, these data imply that galectins recognize damaged Mtb-containing phagosomes, recruit downstream autophagy machinery, and may represent promising targets for host-directed therapeutics to treat Mtb.</jats:p>

Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

“…Second, mutation of OPTN's Ub binding UBAN domain abolishes recruitment to damaged lysosomes and blocks flux. Given that TAX1BP1 can also interact with overexpressed LGALS8 independent of lysosomal damage (Bell et al, 2020;Huttlin et al, 2021) it is formally possible that recruitment can occur via both ubiquitin dependent and independent pathways under some conditions.…”

“…In addition, TAX1BP1 also contains an Nterminal SKICH domain, which interacts with the TBK1-binding adaptor protein NAP1, to facilitate TBK1 binding (Fu et al, 2018). Interestingly, TAX1BP1 has also been recently shown to bind RB1CC1 -a component of the ULK1 kinase complex required for autophagy -in a manner that also requires A114 within the SKICH domain, and a LGALS8 binding element located between residues 632 and 639 (Bell et al, 2020;Ohnstad et al, 2020) (Figure 8A). In order to probe the activities of these various functional elements in lysophagy, we stably expressed various TAX1BP1 mutants in biological triplicate in HeLa TKO cells and performed lysophagic flux assays by flow cytometry after lysosomal damage with LLoMe (Figure 8C, D).…”

Immune evasion and provocation by Mycobacterium tuberculosis

“…Macrophages recognize damaged phagosomes and attempt to route bacteria to the lysosome through selective autophagy. Damaged phagosomes expose glycan residues on intraluminal proteins, which are then bound by galectins, whereas ubiquilin 1 and the ubiquitin ligases parkin and SMURF1 promote ubiquitin deposition around the bacilli 69 , 87 , 88 . Galectin-tagged and ubiquitin-tagged mycobacteria are recognized by adaptor proteins such as p62, NDP52 and TAX1BP1 and are targeted for autolysosomal degradation.…”

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferons by engaging cytosolic DNA sensing in macrophages

“…Therefore, we hypothesized that R. equi may also permeabilize its phagosomal membrane to allow for communication with the host cytosol, resulting in detection by cytosolic DNA sensors. We recently reported that the cytosolic glycanbinding proteins galectin-3, -8, and -9 access the lumen of damaged Mtb-containing vacuoles following ESX-1mediated permeabilization (47). To investigate whether R. equi phagosomal membranes are sufficiently damaged during infection to recruit these galectins, we infected RAW 246.7 cells stably expressing 3xFLAG-tagged galectins (47).…”

“…We recently reported that the cytosolic glycanbinding proteins galectin-3, -8, and -9 access the lumen of damaged Mtb-containing vacuoles following ESX-1mediated permeabilization (47). To investigate whether R. equi phagosomal membranes are sufficiently damaged during infection to recruit these galectins, we infected RAW 246.7 cells stably expressing 3xFLAG-tagged galectins (47). Specifically, we looked at galectins-3, -8, -9 because they have been shown to colocalize with Mtb, L. monocytogenes, Salmonella enterica serovar Typhimurium, and Shigella flexneri (47,48); galectin-1 was For all experiments in this study, statistical significance was determined using Students' t-test.…”