Fig 2: TAX1BP1’s coiled-coil domain and galectin-8’s CRD2 are required for their interaction. (A) Schematic representation of TAX1BP1 domain structure and truncations used in panels B and C. CLIR, noncanonical/LC3C-interacting region; UBZ, ubiquitin-binding zinc finger domain; CC, coiled-coil domains; oligo, oligomerization domain. (B and C) Directed coimmunoprecipitations (IP) of 3×FLAG (FL)-tagged galectin-8 ectopically expressed in HEK293Ts. Whole-cell lysates (WCL) and co-IPs probed for HA-tagged TAX1BP1 truncations. HA-GFP shown as negative control for interaction. (D) Schematic of galectin-8 domain structure and truncations. CRD, carbohydrate recognition domain. (E) Directed co-IPs of HA-TAX1BP1 expressed in HEK293T cells. WCLs and co-IPs probed for FL-galectin-8 truncations. (F) Same as in panels B and C but with mouse (ms) and human (hu) FL-galectin-8, HA-TAX1BP1, and HA-NDP52.

Fig 3: Galectin-8 interacts with exosome-associated proteins and selective autophagy adapter TAX1BP1. (A) Schematic of experimental approach for immunoprecipitation and mass spectrometry (IP-LC/MS) identification of galectin-8 binding partners in macrophages during intracellular bacterial infection. (B) Proteins identified by IP-LC/MS as interacting with galectin-8. (C) Coimmunoprecipitation (IP) of 3×FLAG (FL)-tagged galectins ectopically expressed in HEK293T cells. Whole-cell lysates (WCL) and co-IPs probed for endogenous syntenin-1 and TAX1BP1. (D) WCL and exosomes from FL-galectin-8-expressing RAW 264.7 cells cultured for indicated number of days to assess exosomes accumulated in cell culture media. Endogenous Alix and syntenin-1 were used as markers for exosomes. (E) Directed co-IPs of FL-galectins and HA-tagged selective autophagy adapters expressed ectopically in HEK293T cells. (F) Immunofluorescence of RAW 264.7 cells stably expressing FL-galectin-8 and costained for endogenous TAX1BP1 infected with WT mCherry-expressing Mtb (MOI = 1) 6 h postinfection. Green, endogenous TAX1BP1; magenta, FL-galectin-8; red, mCherry Mtb; blue, DAPI. (G) Quantification of galectin-8+ and TAX1BP1+ Mtb shown in panel F. (H) Same as in panel F but in BMDMs stably expressing GFP–galectin-8. Green, GFP–galectin-8; magenta, endogenous TAX1BP1; red, mCherry Mtb; blue, DAPI. (I) Same as in panel F but in U937s. (J) Immunofluorescence of RAW 264.7 cells infected with ?esat-6 mCherry Mtb and stained for endogenous TAX1BP1 at 6 h postinfection. Green, endogenous TAX1BP1; red, ?esat-6 Mtb; blue, DAPI. Error bars indicate the SD of three coverslips with at least 100 bacteria assessed.

Fig 4: Galectin-8 and TAX1BP1 recognize and target Mtb to selective autophagy in macrophages. Schematic representation of how Mtb is detected by macrophages. Galectin-8 binds to cytosolically exposed glycans in the lumen of damaged Mtb-containing phagosomes. TAX1BP1 is recruited to these damaged phagosomes via its interaction with galectin-8, as well as through its interaction with ubiquitinated substrates. Deletion of galectin-8 results in less targeting of Mtb and increased Mtb survival/replication, while overexpression of galectin-8 leads to increased targeting and less Mtb replication.