EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579.
Bacillus cereus
EsxA
Sporulation
Type VIIb secretion system
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
17 Sep 2024
17 Sep 2024
Historique:
received:
11
01
2024
accepted:
03
09
2024
medline:
18
9
2024
pubmed:
18
9
2024
entrez:
17
9
2024
Statut:
epublish
Résumé
Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished. We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar. Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567. B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.
Sections du résumé
BACKGROUND
BACKGROUND
Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished.
METHODS
METHODS
We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar.
RESULTS
RESULTS
Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567.
CONCLUSION
CONCLUSIONS
B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.
Identifiants
pubmed: 39289639
doi: 10.1186/s12866-024-03492-1
pii: 10.1186/s12866-024-03492-1
doi:
Substances chimiques
Bacterial Proteins
0
Type VII Secretion Systems
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
351Subventions
Organisme : Japan or Japan Society for the Promotion of Science under Grants-in-Aid for Scientific Research
ID : 19K16653
Organisme : Japan Society for the Promotion of Science under Grants-in-Aid for Scientific Research
ID : 21K15430
Organisme : Japan Society for the Promotion of Science under Grants-in-Aid for Scientific Research
ID : 17H01679
Informations de copyright
© 2024. The Author(s).
Références
Acosta Pedemonte NB, Rocchetti NS, Villalba J, Lerman Tenenbaum D, Settecase CJ, Bagilet DH, et al. Bacillus cereus bacteremia in a patient with an abdominal stab wound. Rev Argent Microbiol. 2020;52(2):115–7.
pubmed: 31791818
Akamatsu R, Suzuki M, Okinaka K, Sasahara T, Yamane K, Suzuki S, et al. Novel sequence type in Bacillus cereus strains associated with nosocomial infections and bacteremia, Japan. Emerg Infect Dis. 2019;25(5):883–90.
pubmed: 31002057
pmcid: 6478208
doi: 10.3201/eid2505.171890
Klee SR, Ozel M, Appel B, Boesch C, Ellerbrok H, Jacob D, et al. Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d’Ivoire and Cameroon. J Bacteriol. 2006;188(15):5333–44.
pubmed: 16855222
pmcid: 1540047
doi: 10.1128/JB.00303-06
Enosi Tuipulotu D, Mathur A, Ngo C, Man SM. Bacillus cereus: epidemiology, virulence factors, and host–Pathogen interactions. Trends Microbiol. 2021;29(5):458–71.
pubmed: 33004259
doi: 10.1016/j.tim.2020.09.003
Abdallah AM, van Gey NC, Champion PAD, Cox J, Luirink J, Vandenbroucke-Grauls CMJE, et al. Type VII secretion–mycobacteria show the way. Nat Rev Microbiol. 2007;5(11):883–91.
pubmed: 17922044
doi: 10.1038/nrmicro1773
Bunduc CM, Bitter W, Houben ENG. Structure and function of the mycobacterial type VII Secretion systems. Annu Rev Microbiol. 2020;74(1):315–35.
pubmed: 32660388
doi: 10.1146/annurev-micro-012420-081657
Kneuper H, Cao ZP, Twomey KB, Zoltner M, Jäger F, Cargill JS, et al. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylococcus aureus strains. Mol Microbiol. 2014;93(5):928–43.
pubmed: 25040609
pmcid: 4285178
doi: 10.1111/mmi.12707
Spencer BL, Tak U, Mendonça JC, Nagao PE, Niederweis M, Doran KS. A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence. PLoS Pathog. 2021;17(12):e1010121.
pubmed: 34871327
pmcid: 8675928
doi: 10.1371/journal.ppat.1010121
Pym AS, Brodin P, Brosch R, Huerre M, Cole ST. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol Microbiol. 2002;46(3):709–17.
pubmed: 12410828
doi: 10.1046/j.1365-2958.2002.03237.x
Smith J, Manoranjan J, Pan M, Bohsali A, Xu J, Liu J, et al. Evidence for pore formation in host cell membranes by ESX-1-Secreted ESAT-6 and its role in Mycobacterium marinum escape from the Vacuole. Infect Immun. 2008;76(12):5478–87.
pubmed: 18852239
pmcid: 2583575
doi: 10.1128/IAI.00614-08
Tak U, Dokland T, Niederweis M. Pore-forming esx proteins mediate toxin secretion by Mycobacterium tuberculosis. Nat Commun. 2021;12(1):394.
pubmed: 33452244
pmcid: 7810871
doi: 10.1038/s41467-020-20533-1
Cao Z, Casabona MG, Kneuper H, Chalmers JD, Palmer T. The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. Nat Microbiol. 2016;2(1):16183.
pubmed: 27723728
pmcid: 5325307
doi: 10.1038/nmicrobiol.2016.183
Chan H, Mohamed AMT, Grainge I, Rodrigues CDA. FtsK and SpoIIIE, coordinators of chromosome segregation and envelope remodeling in bacteria. Trends Microbiol. 2022;30(5):480–94.
pubmed: 34728126
doi: 10.1016/j.tim.2021.10.002
Callahan B, Nguyen K, Collins A, Valdes K, Caplow M, Crossman DK, et al. Conservation of structure and protein-protein interactions mediated by the secreted mycobacterial proteins EsxA, EsxB, and EspA. J Bacteriol. 2010;192(1):326–35.
pubmed: 19854905
doi: 10.1128/JB.01032-09
Champion PAD, Stanley SA, Champion MM, Brown EJ, Cox JS. C-Terminal Signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Sci (1979). 2006;313(5793):1632–6.
Warne B, Harkins CP, Harris SR, Vatsiou A, Stanley-Wall N, Parkhill J, et al. The Ess/Type VII secretion system of Staphylococcus aureus shows unexpected genetic diversity. BMC Genomics. 2016;17(1):222.
pubmed: 26969225
pmcid: 4788903
doi: 10.1186/s12864-016-2426-7
Bobrovskyy M, Chen X, Missiakas D. The type 7b secretion system of S. Aureus and its role in colonization and systemic infection. Infect Immun. 2023;91(5).
Klein TA, Grebenc DW, Shah PY, McArthur OD, Dickson BH, Surette MG et al. Dual targeting factors are required for LXG Toxin Export by the bacterial type VIIb secretion system. mBio. 2022;13(5).
Tassinari M, Doan T, Bellinzoni M, Chabalier M, Ben-Assaya M, Martinez M et al. The antibacterial type VII secretion system of Bacillus subtilis: structure and interactions of the pseudokinase YukC/EssB. mBio. 2022;13(5).
Huppert LA, Ramsdell TL, Chase MR, Sarracino DA, Fortune SM, Burton BM. The ESX System in Bacillus subtilis mediates protein secretion. PLoS ONE. 2014;9(5):e96267.
pubmed: 24798022
pmcid: 4010439
doi: 10.1371/journal.pone.0096267
Garufi G, Butler E, Missiakas D. ESAT-6-Like protein secretion in Bacillus anthracis. J Bacteriol. 2008;190(21):7004–11.
pubmed: 18723613
pmcid: 2580693
doi: 10.1128/JB.00458-08
Fleming TC, Shin JY, Lee SH, Becker E, Huang KC, Bustamante C, et al. Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation. Genes Dev. 2010;24(11):1160–72.
pubmed: 20516200
pmcid: 2878653
doi: 10.1101/gad.1925210
Fiche JB, Cattoni DI, Diekmann N, Langerak JM, Clerte C, Royer CA, et al. Recruitment, Assembly, and Molecular Architecture of the SpoIIIE DNA pump revealed by Superresolution Microscopy. PLoS Biol. 2013;11(5):e1001557.
pubmed: 23667326
pmcid: 3646729
doi: 10.1371/journal.pbio.1001557
Rosenberg OS, Dovala D, Li X, Connolly L, Bendebury A, Finer-Moore J, et al. Substrates control multimerization and activation of the multi-domain ATPase motor of type VII secretion. Cell. 2015;161(3):501–12.
pubmed: 25865481
pmcid: 4409929
doi: 10.1016/j.cell.2015.03.040
Nicholson WL, Setlow P, Harwood C, Cutting SM. Molecular biological methods for Bacillus. New York, USA: John Willey; 1990. pp. 391–450.
Plaut RD, Stibitz S. Improvements to a markerless allelic exchange system for Bacillus anthracis. PLoS ONE. 2015;10(12):e0142758.
pubmed: 26624016
pmcid: 4666636
doi: 10.1371/journal.pone.0142758
Gao T, Foulston L, Chai Y, Wang Q, Losick R. Alternative modes of biofilm formation by plant-associated Bacillus cereus. Microbiologyopen. 2015;4(3):452–64.
pubmed: 25828975
pmcid: 4475387
doi: 10.1002/mbo3.251
Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, Kapatral V, et al. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature. 2003;423(6935):87–91.
pubmed: 12721630
doi: 10.1038/nature01582
Simon R, Priefer U, Pühler A. A Broad Host Range Mobilization System for in Vivo Genetic Engineering: Transposon Mutagenesis in Gram negative Bacteria. Bio/Technology. 1983;1(9):784–91.
doi: 10.1038/nbt1183-784
Chan QWT, Howes CG, Foster LJ. Quantitative Comparison of Caste Differences in Honeybee Hemolymph. Mol Cell Proteom. 2006;5(12):2252–62.
doi: 10.1074/mcp.M600197-MCP200
Deng W, Yu HB, de Hoog CL, Stoynov N, Li Y, Foster LJ, et al. Quantitative proteomic analysis of type III secretome of Enteropathogenic Escherichia coli reveals an expanded Effector Repertoire for Attaching/Effacing bacterial pathogens. Cell Proteom. 2012;11(9):692–709. Molecular.
doi: 10.1074/mcp.M111.013672
Qian C, Chen H, Johs A, Lu X, An J, Pierce EM, et al. Quantitative proteomic analysis of biological processes and responses of the Bacterium Desulfovibrio desulfuricans ND132 upon deletion of its Mercury methylation genes. Proteomics. 2018;18(17):1700479.
doi: 10.1002/pmic.201700479
Kanehisa M. Linking databases and organisms: GenomeNet resources in Japan. Trends Biochem Sci. 1997;22(11):442–4.
pubmed: 9397687
doi: 10.1016/S0968-0004(97)01130-4
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–10.
pubmed: 2231712
doi: 10.1016/S0022-2836(05)80360-2
Guy L, Roat Kultima J, Andersson SGE. genoPlotR: comparative gene and genome visualization in R. Bioinformatics. 2010;26(18):2334–5.
pubmed: 20624783
pmcid: 2935412
doi: 10.1093/bioinformatics/btq413
Li L, Jin J, Hu H, Deveau IF, Foley SL, Chen H. Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores. J Ind Microbiol Biotechnol. 2022;49(4).
Huang Q, Zhang Z, Liu Q, Liu F, Liu Y, Zhang J, et al. SpoVG is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in Bacillus cereus. BMC Microbiol. 2021;21(1):0–9.
doi: 10.1186/s12866-021-02239-6
Nicholson WL, Schuerger AC. Bacillus subtilis spore survival and expression of Germination-Induced Bioluminescence after prolonged incubation under simulated Mars Atmospheric pressure and composition: implications for Planetary Protection and Lithopanspermia. Astrobiology. 2005;5(4):536–44.
pubmed: 16078870
doi: 10.1089/ast.2005.5.536
Oktari A, Supriatin Y, Kamal M, Syafrullah H. The bacterial endospore stain on Schaeffer Fulton using variation of Methylene Blue Solution. J Phys Conf Ser. 2017;812:012066.
doi: 10.1088/1742-6596/812/1/012066
Ulhuq FR, Gomes MC, Duggan GM, Guo M, Mendonca C, Buchanan G, et al. A membrane-depolarizing toxin substrate of the Staphylococcus aureus type VII secretion system mediates intraspecies competition. Proc Natl Acad Sci. 2020;117(34):20836–47.
pubmed: 32769205
pmcid: 7456083
doi: 10.1073/pnas.2006110117
Kobayashi K. Diverse LXG toxin and antitoxin systems specifically mediate intraspecies competition in Bacillus subtilis biofilms. PLoS Genet. 2021;17(7):e1009682.
pubmed: 34280190
pmcid: 8321402
doi: 10.1371/journal.pgen.1009682
Poulsen C, Panjikar S, Holton SJ, Wilmanns M, Song YH. WXG100 protein superfamily consists of three subfamilies and exhibits an α-Helical C-Terminal conserved Residue Pattern. PLoS ONE. 2014;9(2):e89313.
pubmed: 24586681
pmcid: 3935865
doi: 10.1371/journal.pone.0089313
Sysoeva TA, Zepeda-Rivera MA, Huppert LA, Burton BM. Dimer recognition and secretion by the ESX secretion system in Bacillus subtilis. Proceedings of the National Academy of Sciences. 2014;111(21):7653–8.
Spencer BL, Doran KS. Evolving understanding of the type VII secretion system in Gram-positive bacteria. PLoS Pathog. 2022;18(7):e1010680.
pubmed: 35901012
pmcid: 9333272
doi: 10.1371/journal.ppat.1010680
Flint JL, Kowalski JC, Karnati PK, Derbyshire KM. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. Proceedings of the National Academy of Sciences. 2004;101(34):12598–603.
Unnikrishnan M, Constantinidou C, Palmer T, Pallen MJ. The enigmatic esx proteins: looking Beyond Mycobacteria. Trends Microbiol. 2017;25(3):192–204.
pubmed: 27894646
doi: 10.1016/j.tim.2016.11.004
Burts ML, Williams WA, DeBord K, Missiakas DM. EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc Natl Acad Sci. 2005;102(4):1169–74.
pubmed: 15657139
pmcid: 545836
doi: 10.1073/pnas.0405620102
Korea CG, Balsamo G, Pezzicoli A, Merakou C, Tavarini S, Bagnoli F, et al. Staphylococcal esx proteins modulate apoptosis and release of Intracellular Staphylococcus aureus during infection in epithelial cells. Infect Immun. 2014;82(10):4144–53.
pubmed: 25047846
pmcid: 4187876
doi: 10.1128/IAI.01576-14
Zhang Q, Wang D, Jiang G, Liu W, Deng Q, Li X, et al. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5. Sci Rep. 2016;6(1):32618.
pubmed: 27600772
pmcid: 5013644
doi: 10.1038/srep32618
Bao Y, Wang L, Sun J. A small protein but with diverse roles: a review of EsxA in Mycobacterium–host Interaction. Cells. 2021;10(7):1645.
pubmed: 34209120
pmcid: 8305481
doi: 10.3390/cells10071645
Renshaw PS, Panagiotidou P, Whelan A, Gordon SV, Hewinson RG, Williamson RA, et al. Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis Complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the Structural properties of ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex. J Biol Chem. 2002;277(24):21598–603.
pubmed: 11940590
doi: 10.1074/jbc.M201625200
Dodson RJ, Durkin AS, Rosovitz MJ, Rasko DA, Hoffmaster A, Ravel J et al. Genome sequence of Bacillus cereus B4264. Vol. Direct sub, NCBI. Maryland; 2008.
Bowman L, Palmer T. The type VII secretion system of Staphylococcus. Annu Rev Microbiol. 2021;75(1):471–94.
pubmed: 34343022
doi: 10.1146/annurev-micro-012721-123600
Bowran K, Palmer T. Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism. Microbiol (N Y). 2021;167(3).
Baron C, Coombes B. Targeting bacterial Secretion systems: benefits of disarmament in the Microcosm. Infect Disord Drug Targets. 2007;7(1):19–27.
pubmed: 17346208
doi: 10.2174/187152607780090685
Belete TM. Novel targets to develop new antibacterial agents and novel alternatives to antibacterial agents. Hum Microb J. 2019;11:100052.
doi: 10.1016/j.humic.2019.01.001
Fyans JK, Bignell D, Loria R, Toth I, Palmer T. The ESX type VII secretion system modulates development, but not virulence, of the plant pathogen Streptomyces scabies. Mol Plant Pathol. 2013;14(2):119–30.
pubmed: 23009676
doi: 10.1111/j.1364-3703.2012.00835.x
Akpe San Roman S, Facey PD, Fernandez-Martinez L, Rodriguez C, Vallin C, Del Sol R, et al. A heterodimer of EsxA and EsxB is involved in sporulation and is secreted by a type VII secretion system in Streptomyces coelicolor. Microbiol (N Y). 2010;156(6):1719–29.
Szklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, et al. The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(D1):D638–46.
pubmed: 36370105
doi: 10.1093/nar/gkac1000
Wu LJ, Lewis PJ, Allmansberger R, Hauser PM, Errington J. A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis. Genes Dev. 1995;9(11):1316–26.
pubmed: 7797072
doi: 10.1101/gad.9.11.1316
Wu LJ, Errington J. Bacillus subtilis spoIIIE protein required for DNA segregation during Asymmetric Cell Division. Sci (1979). 1994;264(5158):572–5.
Eijlander RT, de Jong A, Krawczyk AO, Holsappel S, Kuipers OP. SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis. Nucleic Acids Res. 2014;42(D1):D685–91.
pubmed: 24170806
doi: 10.1093/nar/gkt1007
Higgins D, Dworkin J. Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev. 2012;36(1):131–48.
pubmed: 22091839
doi: 10.1111/j.1574-6976.2011.00310.x
Riley EP, Schwarz C, Derman AI, Lopez-Garrido J. Milestones in Bacillus subtilis sporulation research. Microb Cell. 2021;8(1):1–16.
doi: 10.15698/mic2021.01.739
Omony J, de Jong A, Krawczyk AO, Eijlander RT, Kuipers OP. Dynamic sporulation gene co-expression networks for Bacillus subtilis 168 and the food-borne isolate Bacillus amyloliquefaciens: a transcriptomic model. Microb Genom. 2018;4(2).
Mietrach N, Damián-Aparicio D, Mielich-Süss B, Lopez D, Geibel S. Substrate Interaction with the EssC coupling protein of the type VIIb secretion system. J Bacteriol. 2020;202(7).
Bobrovskyy M, Oh SY, Missiakas D. Contribution of the EssC ATPase to the assembly of the type 7b secretion system in Staphylococcus aureus. J Biol Chem. 2022;298(9):102318.
pubmed: 35921891
pmcid: 9436818
doi: 10.1016/j.jbc.2022.102318