Fig 2: GBP1 Only Targets Already Cytosolic Salmonella and Recruits Caspase-4(A) Representative immunofluorescence images at 2 h p.i. and quantification of the proportion of cytosolic Salmonella Typhimurium (STm) from differentially permeabilized, IFN?-primed THP-1 WT or ?GBP1 cells infected with STm SL1344 (MOI = 30) at indicated time p.i. Cells are outlined by the white, dashed line. Gray: STm; green: pseudo-colored cytosolic and extracellular STm; blue: nuclei. Scale bar, 10 µm.(B) Representative immunofluorescence images at 2 h p.i., and quantification of GBP1 recruitment to cytosolic and intra-vacuolar STm in IFN?-primed and Dox-treated THP-1 ?GBP1+Tet-mCH-GBP1, infected with STm SL1344 (MOI = 30) at indicated times p.i. from differentially permeabilized cells stained for cytosolic STm and total STm. Cells are outlined by the white, dashed line. Red: mCH-GBP1; gray: STm; green: cytosolic STm; blue: nuclei. Scale bar, 10 µm.(C) Quantification of galectin-8 (Gal-8) recruitment to STm SL1344-GFP in IFN?-treated THP-1 WT or ?GBP1 at the indicated times post infection.(D) Representative immunofluorescence images at 1 h and quantification of Gal-8 recruitment to STm in IFN?- and Dox-treated THP-1 ?GBP1+Tet-mCH-GBP1 infected with STm SL1344-GFP (MOI = 30) at the indicated time post infection. Red: mCH-GBP1; gray: STm; green: Gal-8; blue: nuclei. Scale bar, 10 µm.(E) Representative quantification of CellMask fluorescence intensities surrounding STm in infected THP-1 ?GBP1+Tet-mCH-GBP1 pre-treated with IFN? and Dox to induce GBP1 expression at 4 h p.i. plotted depending on whether STm were decorated with GBP1 (+) or not (-). N = number of STm quantified.(F) Fold-change of intracellular STm colony forming units (CFUs) at 18 h p.i. normalized to 2 h p.i. of IFN?-primed THP-1 WT or ?GBP1 cells measured using gentamicin infection-protection assays.Graphs in (A), (B) (C), (D), and (F) show mean ± SEM from n = 3 independent experiments and in (E) representative of n = 2 experiments. *p = 0.05 or ***p = 0.001 in (E) from nested Student’s t test comparing GBP1+ to GBP1- objects from the two experiments following adjustment for multiple comparisons and in (A), (C), and (F) from two-way ANOVA following adjustment for multiple comparisons; ns, not significant.See also Figure S3.

Fig 3: GBP1 Mediates Access to PAMPs During Toxoplasma and Salmonella Infection(A) Left: representative immunofluorescence image of type I Tg grown in human foreskin fibroblasts (HFF WTs) in the presence of EdU DNA label. Labeled type I Tg were harvested from the HFFs and used to infect (inf.) THP-1 ?GBP1+Tet-mCH-GBP1 or THP-1 +myc-AIM2 for 6 h. THP-1 cells were pre-treated with IFN? and Dox to induce mCH-GBP1 expression Middle: parasite DNA released into the cytoplasm was visualized by click-chemistry to label the incorporated EdU. Right: quantification of proportion of infected cells with cytosolic Tg-DNA based grouped based on whether the cells contain a GBP1-targeted (GBP1+) Tg PV. Red: mCH-GBP1 or immune-stained myc-AIM2; white: EdU-DNA; green: Tg; blue: nuclei. Some released, cytosolic (and additionally AIM2-bound, lower panels) Tg-DNA indicated by yellow arrowheads. Scale bar, 10 µm.(B and C) Representative immunofluorescence images (B) and quantification (C) of LPS staining intensity of STm from IFN? and Dox-treated THP-1 ?GBP1+Tet-mCH-GBP1+YFP-CASP4C258S cells infected with STm SL1344 (MOI = 30) for 2 h. Red: mCH-GBP1; gray: STm-LPS; green: YFP-caspase-4; blue: nuclei. Scale bar, 10 µm. Contrast enhanced in the nuclei inset, to visualize STm-DNA used for detection of bacteria that do not stain for LPS.(D) Representative immunofluorescence images at 1 h p.i. and quantification of Gal-8 and caspase-4 (CASP4) recruitment to STm in IFN?-primed and Dox-treated THP-1 ?GBP1+Tet-mCH-GBP1+YFP-CASP4C258S infected with STm SL1344 (MOI = 30) at indicated times p.i. Red: mCH-GBP1; gray: Gal-8; green: YFP-caspase-4; blue: nuclei. Scale bar, 10 µm.(E) Left: silver stain of SDS-PAGE gel showing immunoprecipitation (IP) of FLAG-GBP1 from THP-1 ?GBP1+Tet-FLAG-GBP1 treated with IFN? and Dox and infected with STm for 2 h, left UT. Right: volcano plot of mass spectrometry hits comparing IFN? treated cells with IFN? treated and STm-infected cells. Plotted as Welch difference of mass spectrometry intensities versus -log10(p). Significant hits shown in orange/red circles.(F) Representative immunoblots of IP of endogenous GBP1 from IFN?-primed or naive THP-1 WT infected with STm for 2 h as indicated, showing co-precipitation of endogenous caspase-4 identified as a hit using mass spectrometry and no stable interaction with caspase-1.Graphs in (A) and (D) show mean ± SEM from n = 3 independent experiments. Graph in (C) representative of n = 3 independent experiments. *p = 0.05 and ****p = 0.0001 in (C) from nested one-way ANOVA comparing to undecorated vacuoles for the means of the n = 3 independent experiments; ns, not significant.See also Figure S4 and Data S1.

Fig 4: Damage to endosomal membrane by Ply triggers autophagy mediated clearance of SPN in brain endothelium.A. Intracellular survival efficiency of WT SPN, ?ply mutant and ?ply:pPly strains of R6 (serotype 2) in hBMECs were calculated as percent survival at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA (Tukey’s multiple comparison test). *p<0.05; ***p<0.001. B. Confocal micrographs showing association of Gal8 with GFP expressing WT and ?ply SPN strains at 3 h p.i. in hBMECs. Arrowhead designates bacteria shown in insets. Event localized at z-stack no. 6 and 5 out of 10 and 11 for WT and ?ply, respectively. Scale bar, 5 µm. C. Percent co-localization of Gal8 with WT and ?ply SPN strains at indicated time points post-infection. n = 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate hBMEC cultures. D. Confocal micrograph showing association of WT SPN (blue) with Gal8 (red), NDP52 (pink) and LC3 (green) at 3 h p.i.. hBMECs stably expressing GFP-LC3 were infected with SPN and stained with anti-Gal8 Ab, anti-NDP52 Ab and DAPI. Arrowhead designates bacteria shown in insets. Event localized at z-stack no. 4 out of 9. Scale bar, 5 µm. E. Fluorescent line scan across the yellow line in the merged inset in “D”, depicting co-localization of SPN with Gal8, NDP52 and LC3. F. Quantification of co-localization of LC3 with Gal8 positive SPN in hBMECs at 3 h p.i. Data are presented as mean ± SD of triplicate experiments. n = 50 bacteria per coverslip. G. Western blot demonstrating knock-down in expression of Gal8 in hBMEC cultures following transfection with a targeted siRNA against LGALS8 (siGal8). GAPDH served as loading control. Relative ratio of Gal8 to GAPDH band intensities (normalized to siControl) is mentioned on top of the blot. H. Quantification of LC3 co-localization with WT SPN under Gal8 knockdown conditions using siGal8. Cells transfected with siScrambled served as control (siControl). n = 100 bacteria per coverslip. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using Students t-test. ***p<0.001. I. Intracellular survival of WT SPN in hBMECs following transfection with siGal8 or siControl at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate hBMEC cultures. Statistical analysis was performed using two-way ANOVA (Bonferroni test). ns, non-significant, *p<0.05, **p<0.005. J–L. Transmission electron micrograph depicting WT SPN (J) in vacuolar compartments inside hBMECs. Scale bar, 1 µm. Zoomed in view of the boxed area in “J” and “K” are shown in “K” (scale bar, 0.2 µm) and “L”, respectively. Arrow in “J” points towards SPN sequestered in a vacuole while arrows in “L” show disruptions in the PCV membrane.

Fig 5: Proximity biotinylation of galectins in response to lysosomal damage.(A) Scheme depicting proximity biotinylation of proteins in response to recruitment of LGALS1, LGALS3, and LGALS8 proteins to damaged lysosomes. (B) Experimental workflow for galectin proximity biotinylation. APEX2-tagged LGALS1, LGALS3, and LGALS8 expressed in HeLa cells were subjected to proximity biotinylation 60 min post-LLOMe treatment using 10-plex TMT. (C) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log2 FC versus -Log10 p-value) for APEX-LGALS8-based proximity biotinylation based on the TMT experiment in (B). Specific categories of proteins are indicated by colored circles. (D) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log2 FC versus -Log10 p-value) for APEX-LGALS3-based proximity biotinylation based on the TMT experiment in B. Specific categories of proteins are indicated by colored circles. (E) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log2 FC versus -Log10 p-value) for APEX-LGALS1-based proximity biotinylation based on the TMT experiment in B. Specific categories of proteins are indicated by colored circles. (F) Log2 FC for individual proteins localized to the lysosomal compartment found to be enriched in biotinylated proteins from cells expressing the indicated APEX2-galectin protein. Mean and standard deviation are calculated from two untreated and two treated biological replicates. (G) GO: process enrichment categories for APEX2-LGALS8. Figure 3—source data 1.Log2 FCs for lysosomal proteins from APEX2-LGALS1, 3, and 8 for Figure 3F. Figure 3—source data 2.GO enrichments for APEX2-LGALS8 for Figure 3G.