Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only minor impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.

For Mtb pull-down mass spectrometry, Mtb was fixed in PBS with 4% PFA, washed three times with PBS, and pelleted. 50 μL of Mtb pellet was incubated for 3 hours with 1 mL of 10 mg/mL differentiated THP-1 lysate, washed four times with PBS, and eluted in 8M urea with Rapigest (Waters) before LC-MS sample preparation. Samples were denatured and reduced in 2 M urea, 10 mM NH₄HCO₃, 2 mM DTT for 30 minutes at 60°C, then alkylated with 2 mM iodoacetamide for 45 minutes at room temperature. Trypsin (Promega) was added at a 1:100 enzyme:substrate ratio and digested overnight at 37°C. Following digestion, samples were concentrated using C18 ZipTips (Millipore) according to the manufacturer's specifications. Desalted samples were evaporated to dryness and resuspended in 0.1% formic acid for mass spectrometry analysis.

Peptides were resuspended in 0.1% formic acid and 3% directly injected on a 75 μm ID column (New Objective) packed with 25 cm of Reprosil C18 3 μm, 120 Å particles (Dr. Maisch). Peptides were eluted in positive ion mode into an Orbitrap Elite mass spectrometer (Thermo Fisher) via a Nanospray Flex Ion Source (Thermo Fisher). Elution of peptides was achieved by an acetonitrile gradient delivered at a flow rate of 400 nL/min by an Easy1000 nLC system (Thermo Fisher). All mobile phases contained 0.1% formic acid as buffer A. The total gradient time was 70 minutes, during which mobile phase buffer B (0.1% formic acid in 90% acetonitrile) was ramped from 5% to 30% B over 55 minutes, followed by a ramp to 100% buffer B over a subsequent 5 minutes, and held at 100% for 10 minutes to wash the column. MS data was collected over the first 60 minutes of the gradient. The ion transfer tube was set to 180°C and the spray voltage was 1500V. MS1 scan was collected in the ion trap in centroid mode with 120K resolution over a scan range of 200-2000 m/z, an S-Lens RF of 68%, a 50 ms maximum injection time, and a AGC target of 3e4. Species with a charge state z=1 or unassigned charge states were excluded from selection for MS/MS. Resonance excitation MS/MS was performed on the 20 most abundance precursors with a minimum signal intensity of 200, an isolation width of 2 m/z, a 0.25 activation Q, a 10 ms activation time, a 100 ms maximum injection time, and an AGC target of 1e3. Dynamic exclusion was employed to exclude a list of 50 previously selected precursors within 180 seconds and using a +/- 1.5 Da window. The results raw data was matched to protein sequences as previously described (Penn et al., 2018).