DOI: 10.6078/d1914x
title: Mass spectrometry data from: Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens

description: 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.
description: For <em>Mtb</em> pull-down mass
 spectrometry, <em>Mtb </em>was fixed in PBS with 4% PFA,
 washed three times with PBS, and pelleted. 50 μL
 of <em>Mtb </em>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
 NH<sub>4</sub>HCO<sub>3</sub>, 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).