Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion

Abstract

Autophagy defends the mammalian cytosol against bacterial infection. Efficient pathogen engulfment is mediated by cargo-selecting autophagy adaptors that rely on unidentified pattern-recognition or danger receptors to label invading pathogens as autophagy cargo, typically by polyubiquitin coating. Here we show in human cells that galectin 8 (also known as LGALS8), a cytosolic lectin, is a danger receptor that restricts Salmonella proliferation. Galectin 8 monitors endosomal and lysosomal integrity and detects bacterial invasion by binding host glycans exposed on damaged Salmonella-containing vacuoles. By recruiting NDP52 (also known as CALCOCO2), galectin 8 activates antibacterial autophagy. Galectin-8-dependent recruitment of NDP52 to Salmonella-containing vesicles is transient and followed by ubiquitin-dependent NDP52 recruitment. Because galectin 8 also detects sterile damage to endosomes or lysosomes, as well as invasion by Listeria or Shigella, we suggest that galectin 8 serves as a versatile receptor for vesicle-damaging pathogens. Our results illustrate how cells deploy the danger receptor galectin 8 to combat infection by monitoring endosomal and lysosomal integrity on the basis of the specific lack of complex carbohydrates in the cytosol.

Figure 1. Galectin-8 responds to infection by S.Typhimurium and restricts bacterial proliferation

A-B) Analysis of HeLa cells stably expressing YFP fused to the indicated galectins and infected with S.Typhimurium for 1h. A) Percentage of bacteria coated by the indicated galectins. YFP-positive bacteria were counted by microscopy. Mean and s.d. of triplicate HeLa cultures, n>100 bacteria per coverslip. B) Confocal micrographs. Arrowheads, bacteria shown in insets. C) Kinetics of fold replication for S.Typhimurium in HeLa cells transfected with the indicated siRNAs. Bacteria were counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts. siRNAs are further characterized in Supplementary Figure 2a-c. ** P<0.01, Student’s t-test. Scale bar 10 μm.

Figure 2. Galectin-8 binds NDP52

A) LUMIER binding assay: Normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing NDP52, p62, or Optineurin each fused to luciferase, and the indicated Flag-tagged galectins were incubated with anti-Flag beads. Flag-tagged proteins are further characterized in Supplementary Figure S4a. B) Lysates of 293ET cells, expressing Flag-tagged proteins as indicated, were immunoprecipitated with anti-Flag beads. Lysates and precipitates (IP) were blotted for the presence of Flag-tagged proteins and endogenous NDP52. C) Confocal images of HeLa cells infected with S.Typhimurium for 1h and stained with antisera against NDP52 and galectin-8. Arrowheads, bacteria shown in insets. D) Colocalization of NDP52 with galectin-positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins, infected with S.Typhimurium and stained with NDP52 antiserum 1h after infection. Mean and s.d. of duplicate HeLa cultures, n>100 bacteria per coverslip, representative of 2 independent experiments. Scale bar 10 μm.

Figure 3. Galectin-8 is a danger receptor that senses cytosolic host glycans and recruits NDP52 to restrict Salmonella proliferation.

A) Percentage of S.Typhimurium coated by the indicated galectins. HeLa cells stably expressing YFP-tagged galectins were treated with the indicated siRNAs. YFP-positive bacteria were counted by microscopy at 1h post infection. siRNAs are further characterized in Supplementary Figure 2. B) Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum. NDP52-positive bacteria were counted by microscopy 1h after infection with S.Typhimurium. siRNAs are further characterized in Supplementary Figure 2. C) Percentage of bacteria coated by the indicated galectin-8 alleles. HeLa cells stably expressing the indicated galectin-8 alleles fused to YFP were infected with S.Typhimurium. YFP-positive S.Typhimurium were counted by microscopy at 75min p.i. D) Binding of galectin-8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST-ubiquitin, His-GST-galectin-8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum. E) Percentage of S.Typhimurium coated by the indicated galectins. Wild type CHO cells and mutant Lec3.2.8.1 cells stably expressing YFP-tagged galectins were infected with S.Typimurium. YFP-positive bacteria were counted by microscopy at 1h post infection. F) Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without PEG as indicated, followed by hypotonic shock. G) Fold replication of S.Typhimurium in HeLa cells expressing the indicated galectin-3 variants and transfected with the indicated siRNAs. At 2 and 6h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin-3 proteins are further characterized in Supplementary Figure 4b. Mean and s.d. of duplicate coverslips (A-C,E) or triplicate HeLa cultures and duplicate colony counts (G). >100 bacteria counted per coverslip. Data are representative of at least two repeats. * P<0.05, Student’s t-test. Scale bar 10 μm

Figure 4. The anti-bacterial effect of galectin-8 is mediated by autophagy

A) Confocal micrograph of HeLa cells expressing GFP-LC3 and mCherry-galectin-8, stained for NDP52 1h after infection with S.Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets. B) Percentage of GFP-LC3 positive S.Typhimurium at 1h p.i. in HeLa cells treated with the indicated siRNAs. C) Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S.Typhimurium. D) Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP-galectin-8 as indicated, were infected with S.Typhimurium. Ubiquitin was detected by antibody staining. E) HeLa cells treated with the indicated siRNAs were infected with S.Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. * P<0.05, Student’s t-test, scale bar 10 μm.