A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection

  1. David B Heisler
  2. Kristen A Johnson
  3. Duo H Ma
  4. Maikke B Ohlson
  5. Lishu Zhang
  6. Michelle Tran
  7. Chase D Corley
  8. Michael E Abrams
  9. Jeffrey G McDonald
  10. John W Schoggins
  11. Neal M Alto  Is a corresponding author
  12. Arun Radhakrishnan  Is a corresponding author
  1. Department of Microbiology, The University of Texas Southwestern Medical Center, United States
  2. Department of Molecular Genetics, The University of Texas Southwestern Medical Center, United States
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 5 supplements
Comparison of oxysterol specificities for effects on PM cholesterol pools, stimulation of ACAT activity, and suppression of SREBP-2 cleavage.

(A, B) Time course and dose curve analysis of 25HC treatment. On day 0, CHO-K1 cells were set up in medium B at a density of 6x104 cells per well of a 48-well plate. On day 1, media was removed, …

Figure 1—figure supplement 1
Comparison of effects of oxysterols on PM cholesterol pools – immunoblot analysis.

(A) Schematic of protein sensors used to monitor changes in PM cholesterol pools. (B, C) Quantification of immunoblot analysis shown in Figure 1D and E. The intensities of bands corresponding to …

Figure 1—figure supplement 2
Comparison of effects of oxysterols on PM cholesterol pools – fluorescence microscopy analysis.

On day 0, CHO-K1 cells were set up in medium B at a density of 3x104 cells per well of an 8-well Lab-Tek II chambered #1.5 coverglass dish. On day 1, media was removed, cells were washed twice with …

Figure 1—figure supplement 3
Comparison of effects of oxysterols on accessible PM cholesterol levels – immunoblot analysis.

On day 0, CHO-K1 cells were set up in medium B at a density of 6x104 cells per well of a 48-well plate. On day 1, media was removed, cells were washed twice with 500 µl of PBS followed by addition …

Figure 1—figure supplement 4
Treatment of red blood cells with oxysterols does not affect levels of accessible cholesterol in their membranes.

(A, B) Each binding assay contained 500 µl of rabbit red blood cells (RBCs) that had been washed and diluted as described in Materials and methods. RBCs were then subjected to one of the following …

Figure 1—figure supplement 5
Quantification of immunoblot analysis shown in Figure 1G.

The intensities of bands corresponding to nuclear SREBP2 in Figure 1G were quantified by densitometry and normalized to cellular actin levels as described in Materials and methods. The intensity …

Figure 2 with 5 supplements
25HC fails to trigger rapid depletion of accessible cholesterol from PMs of ACAT-deficient cells.

(A) Immunoblot analysis of ALOD4 binding. On day 0, the indicated versions of CHO-K1 cells were set up in medium B at a density of 6x104 cells per well of a 48-well plate. On day 1, media was …

Figure 2—figure supplement 1
Characterization of a CHO-K1 cell line deficient in ACAT1.

(A) Strategy for generating ACAT1-deficient CHO-K1 cells using CRISPR-Cas9 technology. Two guide RNAs were designed to target and disrupt exons 2 and 14 of hamster ACAT1 (also designated as Soat1). …

Figure 2—figure supplement 2
Quantification of immunoblot analysis shown in Figure 2A.

The intensities of bands corresponding to bound His6-FLAG-ALOD4 in Figure 2A were quantified by densitometry and normalized to cellular actin levels as described in Materials and Methods. The …

Figure 2—figure supplement 3
Effects of 25HC on SREBP2 processing in ACAT-deficient cells.

On day 0, the indicated versions of CHO-K1 cells were set up in medium B at a density of 6x104 cells per well of a 48-well plate. On day 1, media was removed, cells were washed twice with 500 µl of …

Figure 2—figure supplement 4
Oxysterols that activate ACAT fail to trigger rapid depletion of accessible cholesterol from PMs of ACAT-deficient cells.

On day 0, the indicated versions of CHO-K1 cells were set up in medium B at a density of 6x104 cells per well of a 48-well plate. On day 1, media was removed, followed by addition of 200 µl of …

Figure 2—figure supplement 5
Quantification of immunoblot analysis shown in Figure 2C and D.

(A, B) The intensities of bands corresponding to bound His6-FLAG-ALOD4 (left panels of Figure 2C and D) and bound OlyA-His6 (right panels of Figure 2C and D) were quantified by densitometry and …

Figure 3 with 1 supplement
Intracellular cholesterol trafficking and steady state levels of PM accessible cholesterol in ACAT-deficient cells.

On day 0, wild-type and ACAT1-deficient CHO-K1 cells were set up in medium B at a density of 5x104 cells per well of a 24-well plate (A, B) or 6x104 cells per well of a 48-well plate (C - E). (A, B) …

Figure 3—figure supplement 1
Quantification of immunoblot analysis shown in Figure 3C, D and E.

(A – C) The intensities of bands corresponding to precursor (P) and nuclear (N) forms of SREBP2 in Figure 3C, D and E were quantified by densitometry and these values were used to calculate the …

Figure 4 with 2 supplements
25HC-triggered depletion of accessible cholesterol from PMs persists for long times through suppression of SREBP-mediated cholesterol synthesis and uptake, and continued activation of ACAT.

(A) Retention of 25HC in cells prevents replenishment of accessible cholesterol on PMs after it has been depleted by 25HC. On day 0, CHO-K1 cells were set up in medium B in eight 24-well plates at a …

Figure 4—figure supplement 1
Recovery of accessible cholesterol on PMs after depletion by HPCD.

On day 0, CHO-K1 cells were set up in medium B at a density of 1.5x105 cells per well of a 24-well plate. On day 1, media was removed, cells were washed twice with 1 ml of PBS followed by addition …

Figure 4—figure supplement 2
Quantification of immunoblot analysis shown in Figure 4B, C and D.

The intensities of bands corresponding to bound His6-FLAG-ALOD4 in Figure 4B, C and D were quantified by densitometry and normalized to cellular actin levels as described in Materials and methods. …

Figure 5 with 1 supplement
25HC fails to protect ACAT-deficient cells from pore formation by bacterial cytolysins.

On day 0, the indicated versions of CHO-K1 cells were set up in medium B at a density of 7.5x104 cells per well of a 48-well plate. On day 1, media was removed and replaced with 500 µl of medium B …

Figure 5—figure supplement 1
Susceptibility of wild-type and ACAT-deficient CHO-K1 cells to pore formation by bacterial cytolysins.

(A, B) Pore formation by ALO and PFO. On day 0, the indicated versions of CHO-K1 cells were set up in medium B at a density of 7.5x104 cells per well of a 48-well plate. On day 1, media was removed, …

Figure 6 with 1 supplement
25HC fails to protect ACAT-deficient cell lines and mice from infection by Listeria monocytogenes.

(A) Listeria infection of CHO-K1 cells. On day 0, the indicated versions of CHO-K1 cells were set up in medium I at a density of 1x105 cells per well of a 24-well plate. On day 1, media was …

Figure 6—figure supplement 1
Susceptibility of wild-type and ACAT-deficient CHO-K1 cells to infection by Listeria monocytogenes.

On day 0, the indicated versions of CHO-K1 cells were set up in medium I at a density of 1x105 cells per well of a 24-well plate. On day 1, cells were infected with Listeria monocytogenes (MOI = 1) …

Figure 7 with 1 supplement
25HC fails to protect ACAT-deficient cells from viral infection.

(A, B) Viral infection of Huh7.5 cells. On day 0, the indicated versions of Huh7.5 cells were set up in medium E at a density of 7x104 cells per well of a 24-well plate. On day 1, media was removed …

Figure 7—figure supplement 1
Characterization of a Huh7.5 cell line deficient in ACAT1 and ACAT2.

(A) Strategy for generating Huh7.5 cells deficient in ACAT1 and ACAT2. Guide RNAs were designed to target and disrupt exon 6 in human ACAT1 (also designated as SOAT1) and exon 2 in human ACAT2 (also …

Author response image 1
Inhibition of ACAT activity and depletion of accessible PM cholesterol by 25HC in RAW macrophage cells.

(A) ACAT activity [same procedure as in Figure 1F]. On day 0, cells were set up either in medium D (RAW) or medium B (CHO-K1) at a density of 2.5 x 105 cells per 60-mm dish. One day 2, media was …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Cricetulus griseus)Soat1NCBI GeneGene ID:100689317
Gene (H. sapiens)SOAT1NCBI GeneGene ID:6646
Gene (H. sapiens)SOAT2NCBI GeneGene ID:8435
Strain, strain background (Mus musculus)C57BL/6 JThe Jackson LaboratoryCat#000664
Strain, strain background (M. musculus)Wild Type C57BL/6JSoat1+/+This paperExperimental Wild-type mice
Strain, strain background (M. musculus)Mouse: ACAT1+/-: C57BL/6JSoat+/-This paperHeterozygous ACAT1 mice
Strain, strain background (M. musculus)Mouse: ACAT1-/-: C57BL/6JSoat1-/-This paperPMID:8943057Experimental ACAT1 KO mice
Strain, strain background (Listeria monocytogenes)10403sPMID:3114382Gift from Dr. Daniel Portnoy, University of California, Berkeley
Strain, strain background (L. monocytogenes)EGD InlAmutPMID:17540170Gift from Dr. Wolf-Dieter Schubert, University of Pretoria, South Africa
Strain, strain background (Escherichia coil)BL21 (DE3) pLysSInvitrogenCat#C606003Chemically competent cells
Strain, strain background (Coronaviridae)hCoV-OC43ATCCCat# VR-1558
Cell line (C. griseus)CHO-K1ATCCCAT#CCL-61
Cell line (C. griseus)CHO-K1 ACAT1 KOThis paperDr. Arun Radhakrishnan (UTSW)
Cell line (C. griseus)CHO-K1 ACAT1 KO; hACAT1(WT)This paperDr. Arun Radhakrishnan (UTSW)
Cell line (C. griseus)CHO-K1 ACAT1 KO; hACAT1(H460A)This paperDr. Arun Radhakrishnan (UTSW)
Cell line (C. griseus)M19PMID:9748295Dr. Arun Radhakrishnan (UTSW)
Cell line (C. griseus)SRD13APMID:10497220Dr. Arun Radhakrishnan (UTSW)
Cell line (H. sapiens)HEK293APMID:32284563Dr. Neal Alto (UTSW)
Cell line (H. sapiens)HEK293A LXRα;LXRβ-deficient cellsPMID:32284563Dr. Neal Alto (UTSW)
Cell line (H. sapiens)HEK293TPMID:21478870Dr. John Schoggins (UTSW)
Cell line (H. sapiens)Huh7.5PMID:21478870Dr. John Schoggins (UTSW)
Cell line (H. sapiens)Huh7.5ΔACATThis paperDr. Neal Alto (UTSW)
Transfected construct (human)pCDNA6.ZikaMR766.Venus3115Intron HDVrPMID:27704051Dr. Matthew Evans (Icahn School of Medicine at Mount Sinai)
Transfected construct (human)pGag-polOtherDr. Charles Rice (Rockefeller University)
Transfected construct (human)pLentiCRISPRv2-BlastAddgeneCat#98293
Transfected construct (human)pLentiCRISPRv2-PuroAddgeneCat#98290
Transfected construct (human)pTRIP.CMV.IVSB.ires.TagRFPPMID:21478870Dr. John Schoggins (UTSW)
Transfected construct (human)pTRIP.CMV.hACAT1.ires.TagRFPThis paperDr. Arun Radhakrishnan (UTSW)
Transfected construct (human)pTRIP.CMV.hACAT1(H460A).ires.TagRFPThis paperDr. Arun Radhakrishnan (UTSW)
Transfected construct (human)pVSV-GlycoproteinOtherDr. Charles Rice (Rockefeller University)
Antibodyanti-mouse IgG (H+L) (Donkey Polyclonal Peroxidase-AffiniPure)Jackson ImmunoResearchCat#715-035-150; RRID: AB_2340770WB (1:5,000)
Antibodyanti-rabbit IgG (H+L) (Goat Polyclonal Peroxidase AffiniPure)Jackson ImmunoResearchCat#111-035-003; RRID:AB_2313567WB (1:5,000)
Antibodyanti-Coronavirus Group Antigen, nucleoprotein of OC-43, 229E strain, clone 542-7D (Mouse Monoclonal)Millipore SigmaCat# MAB9013; RRID:AB_95425Flow cytometry (1:50)
Antibodyanti-FLAG M2 clone (Mouse Monoclonal)Sigma-AldrichF1804; RRID:AB_262044WB (1:1,000)
Antibodyanti-His Tag, clone HIS.H8 (Mouse Monoclonal)MilliporeCat#05–949; RRID:AB_492660WB (1:1,000)
Antibodyanti-SREBP2 (Mouse Monoclonal)Ref. (95)IgG-7D4WB (10 μg/ml)
Antibodyanti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Mouse Polyclonal Goat)Thermo Fisher ScientificCat#A-11001; RRID:AB_2534069Flow cytometry (1:1,000)
Antibodyanti-ACAT1 (Rabbit Polyclonal)NovusCat#NB400-141; RRID:AB_10001588WB (1:1,000)
Recombinant DNA reagentHis6-ALO(FL) in pRSET-BPMID:25809258Dr. Arun Radhakrishnan (UTSW)
Recombinant DNA reagentHis6-FLAG-ALOD4 in pRSET-BPMID:33712199Dr. Arun Radhakrishnan (UTSW)
Recombinant DNA reagentHis6-Neon-FLAG-ALOD4 in pRSET-BPMID:33712199Dr. Arun Radhakrishnan (UTSW)
Recombinant DNA reagentOlyA-His6 in pRSET-BPMID:33712199Dr. Arun Radhakrishnan (UTSW)
Recombinant DNA reagentHis6-PFO(FL) in pRSET-BPMID:25809258Dr. Arun Radhakrishnan (UTSW)
Sequence-based reagentCHO-K1 Soat1 gRNA for Exon 2This paperTarget sequenceAGGAACCGGCTGTCAAAATC
Sequence-based reagentCHO-K1 Soat1 gRNA for Exon 14This paperTarget sequenceATAGCTCAAGCAGACAGCGA
Sequence-based reagentHuh7.5 SOAT1 gRNA for Exon 6This paperTarget sequenceCACCAGGTCCAAACAACGGT
Sequence-based reagentHuh7.5 SOAT2 gRNA for Exon 2This paperTarget sequenceGGTCCATTGTACCAAGTCCG
Sequence-based reagentCHO-K1 Soat1 Exon 2 ForwardThis paperPCR PrimerCTACAAGAGCTAGTTTCAGG
Sequence-based reagentCHO-K1 Soat1 Exon 2 ReverseThis paperPCR PrimerCCCTGTGTGTACAGTGCCTT
Sequence-based reagentCHO-K1 Soat1 Exon 14 ForwardThis paperPCR PrimerTCACTCACCTTGAAGACCCA
Sequence-based reagentCHO-K1 Soat1 Exon 14 ReverseThis paperPCR PrimerGGGTTCCTCTCTACACACTCA
Sequence-based reagentHuh7.5 Soat1 Exon 6 ForwardThis paperPCR PrimerCAGCGTATTAACGTTGTGGTGT
Sequence-based reagentHuh7.5 Soat1 Exon 6 ReverseThis paperPCR PrimerGCCCAATGTTGAAACAGAAAAT
Sequence-based reagentHuh7.5 Soat2 Exon 2 ForwardThis paperPCR PrimerCAACTTCCCCTTCTAGTAGCCC
Sequence-based reagentHuh7.5 Soat2 Exon 2 ReverseThis paperPCR PrimerCTTTATCACCAAGCCTCACTCC
Commercial assay or kitCytofix/Cytoperm Fixation/Permeabilization KitBD BiosciencesCat#554714;
RRID:AB_2869008
Commercial assay or kitLookOut Mycoplasma PCR Detection KitMillipore SigmaCat# MP0035
Commercial assay or kitMicroplate BCA Protein Assay KitThermo Fisher ScientificCat#23252
Chemical compound, drug19-hydroxycholesterolSteraloidsCat#C6470-000
Chemical compound, drug2-Hydroxypropyl-β-cyclodextrin (HPCD)Cyclodextrin Technologies
Development
Cat#THPB-P
Chemical compound, drug20(R)-hydroxycholesterolAvanti Polar LipidsCat#700156
Chemical compound, drug22(R)-hydroxycholesterolAvanti Polar LipidsCat#700058
Chemical compound, drug22(S)-hydroxycholesterolAvanti Polar LipidsCat#700057
Chemical compound, drug24(R)-hydroxycholesterolAvanti Polar LipidsCat#700071
Chemical compound, drug24(S)-hydroxycholesterolAvanti Polar LipidsCat#700061
Chemical compound, drug25-hydroxycholesterolAvanti Polar LipidsCat#700019
Chemical compound, drug25-hydroxycholesterol-3-sulfateAvanti Polar LipidsCat#700017
Chemical compound, drug25-hydroxycholesterol-d6Avanti Polar LipidsCat#700053
Chemical compound, drug27-hydroxycholesterolAvanti Polar LipidsCat#700021
Chemical compound, drug4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI)Millipore SigmaCat#D8417Microscopy (1 µg/ml)
Chemical compound, drug4β-hydroxycholesterolAvanti Polar LipidsCat#700036
Chemical compound, drug7α-hydroxycholesterolAvanti Polar LipidsCat#700034
Chemical compound, drugAccumaxInnovative Cell TechnologiesCat#AM105
Chemical compound, drugAlexa Fluor 647 C2-maleimideThermo Fisher ScientificCat#A20347
Chemical compound, drugBlasticidin S HClThermo Fisher ScientificCat#A1113903
Chemical compound, drugBovine serum albuminMillipore SigmaCat#P0834
Chemical compound, drugBrain Heart Infusion AgarThermo Fisher ScientificCat#DF0418
Chemical compound, drugBrain Heart Infusion BrothThermo Fisher ScientificCat#DF0037
Chemical compound, drugCholesterolMillipore SigmaCat#C8667
Chemical compound, drugcOmplete, EDTA-free Protease Inhibitor CocktailRocheCat# 05056489001
Chemical compound, drugDulbecco’s Modified Eagle Medium (DMEM) – high glucoseSigmaCat#D6429
Chemical compound, drugDulbecco’s Modified Eagle Medium (DMEM) – low glucoseSigmaCat#D6046
Chemical compound, drugDulbecco’s Modified Eagle Medium (DMEM)/F-12 (1:1 mixture)CorningCat#10–090-CV
Chemical compound, drugDulbecco’s phosphate buffered saline (PBS)Thermo Fisher ScientificCat#MT21031CV
Chemical compound, drugFetal Calf SerumSigma-AldrichCat#F2442
Chemical compound, drugFluorescein-5-maleimideThermo Fisher ScientificCat#62245
Chemical compound, drugGentamicinQuality BiologicalCat#120-098-661
Chemical compound, drugGhost Dye Violet 450Tonbo biosciencesCat#13–0863
Chemical compound, drugHanks Balanced Salt Solution (HBSS)Millipore SigmaCat#14175–095
Chemical compound, drugHexadimethrine bromideThermo Fisher ScientificCat#107689
Chemical compound, drugMEM Non-Essential Amino Acids Solution (100 X)Thermo Fisher ScientificCat#11140050
Chemical compound, drugMethyl-β-cyclodextrin, randomly methylated (MCD)Cyclodextrin Technologies DevelopmentCat#TRMB-P
Chemical compound, drugMuristerone ASigma-AldrichCat#M7888
Chemical compound, drugOpti-MEMThermo Fisher ScientificCat#31985062
Chemical compound, drugParaformaldehydeAlfa AesarCat#43368
Chemical compound, drugPenicillin/StreptomycinGibcoCat#15140–122
Chemical compound, drugPhenylmethylsulfonyl fluoride (PMSF)GoldbioCat#P-470–25
Chemical compound, drugPuromycinThermo Fisher ScientificCat#A1113803
Chemical compound, drugRabbit Whole BloodInnovative ResearchCat#
IGRBWBK2E10ML
Chemical compound, drugSodium compactinPMID:624722N/A
Chemical compound, drugSodium mevalonatePMID:624722N/A
Chemical compound, drugSandoz 58–035 (SZ58-035)Millipore SigmaCat#S9318
Chemical compound, drugTris (2-carboxyethyl) phosphine Hydrochloride (TCEP)GoldbioCat#TCEP1
Software, algorithmBenchling CRISPR Guide RNA design toolBenchlinghttps://www.benchling.com/crispr
Software, algorithmCHOP-CHOPPMID:31106371https://chopchop.cbu.uib.no/
Software, algorithmFlowJoBD (Becton, Dickinson & Co.)http://www.flowjo.com
Software, algorithmImageJPMID:22930834https://imagej.nih.gov/ij/
OtherEclipse Ti epifluorescence microscopeNikon Inc.N/AInstrument used for microscopy imaging studies.
OtherS1000 Flow CytometerStratedigm Inc.NAInstrument used for flow cytometry analysis.

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