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Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence


Autoři: Jamie L. Burgess aff001;  Heather B. Case aff001;  R. Alan Burgess aff001;  Nicholas E. Dickenson aff001
Působiště autorů: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah, United States of America aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0228227

Souhrn

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo.

Klíčová slova:

Adenosine triphosphatase – Fluorescence microscopy – Oligomers – Protein secretion – Secretion systems – Shigella – Shigella flexneri – ATP hydrolysis


Zdroje

1. Burkinshaw BJ, Strynadka NCJ. Assembly and structure of the T3SS. Biochimica Et Biophysica Acta. 2014;1843(8):1649–63. doi: 10.1016/j.bbamcr.2014.01.035 24512838

2. Carayol N, Tran Van Nhieu G. The inside story of Shigella invasion of intestinal epithelial cells. Cold Spring Harb Perspect Med. 2013;3(10):a016717. doi: 10.1101/cshperspect.a016717 24086068.

3. Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion. Clin Microbiol Rev. 2008;21(1):134–56. doi: 10.1128/CMR.00032-07 18202440.

4. Puhar A, Sansonetti PJ. Type III secretion system. Curr Biol. 2014;24(17):R784–91. doi: 10.1016/j.cub.2014.07.016 25202865.

5. Ashida H, Mimuro H, Sasakawa C. Shigella manipulates host immune responses by delivering effector proteins with specific roles. Front Immunol. 2015;6:219. doi: 10.3389/fimmu.2015.00219 25999954.

6. Enninga J, Mounier J, Sansonetti P, Tran Van Nhieu G. Secretion of type III effectors into host cells in real time. Nat Methods. 2005;2(12):959–65. doi: 10.1038/nmeth804 16299482.

7. Portaliou AG, Tsolis KC, Loos MS, Zorzini V, Economou A. Type III Secretion: Building and Operating a Remarkable Nanomachine. Trends in Biochemical Sciences. 2016;41(2):175–89. doi: 10.1016/j.tibs.2015.09.005 26520801.

8. Galan JE, Lara-Tejero M, Marlovits TC, Wagner S. Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. Annu Rev Microbiol. 2014;68:415–38. doi: 10.1146/annurev-micro-092412-155725 25002086.

9. Cossart P, Sansonetti PJ. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science (New York, NY). 2004;304(5668):242–8. doi: 10.1126/science.1090124 15073367.

10. Chatterjee S, Chaudhury S, McShan AC, Kaur K, De Guzman RN. Structure and biophysics of type III secretion in bacteria. Biochemistry. 2013;52(15):2508–17. doi: 10.1021/bi400160a 23521714

11. Schroeder GN, Jann NJ, Hilbi H. Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death. Microbiology. 2007;153(Pt 9):2862–76. doi: 10.1099/mic.0.2007/007427-0 17768231.

12. Adam PR, Dickenson NE, Greenwood JC 2nd, Picking WL, Picking WD. Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes. Proteins. 2014;82(11):3013–22. doi: 10.1002/prot.24662 25103195.

13. Dickenson NE, Picking WD. Forster resonance energy transfer (FRET) as a tool for dissecting the molecular mechanisms for maturation of the Shigella type III secretion needle tip complex. Int J Mol Sci. 2012;13(11):15137–61. doi: 10.3390/ijms131115137 23203116.

14. Hu B, Morado DR, Margolin W, Rohde JR, Arizmendi O, Picking WL, et al. Visualization of the type III secretion sorting platform of Shigella flexneri. Proceedings of the National Academy of Sciences of the United States of America. 2015;112(4):1047–52. doi: 10.1073/pnas.1411610112 25583506.

15. Akeda Y, Galan JE. Chaperone release and unfolding of substrates in type III secretion. Nature. 2005;437(7060):911–5. doi: 10.1038/nature03992 16208377.

16. Allison SE, Tuinema BR, Everson ES, Sugiman-Marangos S, Zhang K, Junop MS, et al. Identification of the docking site between a type III secretion system ATPase and a chaperone for effector cargo. J Biol Chem. 2014;289(34):23734–44. doi: 10.1074/jbc.M114.578476 25035427.

17. Erhardt M, Mertens ME, Fabiani FD, Hughes KT. ATPase-independent type-III protein secretion in Salmonella enterica. PLoS Genet. 2014;10(11):e1004800. doi: 10.1371/journal.pgen.1004800 25393010.

18. Minamino T, Morimoto YV, Hara N, Aldridge PD, Namba K. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export. PLoS Pathog. 2016;12(3):e1005495. doi: 10.1371/journal.ppat.1005495 26943926.

19. Minamino T, Namba K. Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature. 2008;451(7177):485–8. doi: 10.1038/nature06449 18216858.

20. Wilharm G, Lehmann V, Krauss K, Lehnert B, Richter S, Ruckdeschel K, et al. Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB. Infect Immun. 2004;72(7):4004–9. doi: 10.1128/IAI.72.7.4004-4009.2004 15213145.

21. Burgess JL, Burgess RA, Morales Y, Bouvang JM, Johnson SJ, Dickenson NE. Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation. J Biol Chem. 2016;291(50):25837–52. doi: 10.1074/jbc.M116.755256 27770024.

22. Burgess JL, Jones HB, Kumar P, Toth RT, Middaugh CR, Antony E, et al. Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri. Protein Science: A Publication of the Protein Society. 2016;25(5):1037–48. doi: 10.1002/pro.2917 26947936.

23. Blaylock B, Riordan KE, Missiakas DM, Schneewind O. Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL. Journal of Bacteriology. 2006;188(10):3525–34. doi: 10.1128/JB.188.10.3525-3534.2006 16672607

24. Andrade A, Pardo JP, Espinosa N, Pérez-Hernández G, González-Pedrajo B. Enzymatic characterization of the enteropathogenic Escherichia coli type III secretion ATPase EscN. Archives of Biochemistry and Biophysics. 2007;468(1):121–7. doi: 10.1016/j.abb.2007.09.020 17964526

25. Stone CB, Johnson DL, Bulir DC, Gilchrist JD, Mahony JB. Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae. Journal of Bacteriology. 2008;190(20):6580–8. doi: 10.1128/JB.00761-08 18708502

26. Akeda Y, Galan JE. Genetic analysis of the Salmonella enterica type III secretion-associated ATPase InvC defines discrete functional domains. J Bacteriol. 2004;186(8):2402–12. doi: 10.1128/JB.186.8.2402-2412.2004 15060043.

27. Formal SB, Dammin GJ, Labrec EH, Schneider H. Experimental Shigella infections: characteristics of a fatal infection produced in guinea pigs. Journal of Bacteriology. 1958;75(5):604–10. 13538931.

28. Jouihri N, Sory MP, Page AL, Gounon P, Parsot C, Allaoui A. MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI, but not of Ipa proteins, through the type III secretion apparatus of Shigella flexneri. Mol Microbiol. 2003;49(3):755–67. doi: 10.1046/j.1365-2958.2003.03590.x 12864857.

29. Case HB, Mattock DS, Dickenson NE. Shutting Down Shigella Secretion: Characterizing Small Molecule Type Three Secretion System ATPase Inhibitors. Biochemistry. 2018;57(50):6906–16. doi: 10.1021/acs.biochem.8b01077 30460850.

30. Case HB, Dickenson NE. MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47. Biochemistry. 2018;57(15):2266–77. doi: 10.1021/acs.biochem.8b00070 29595954.

31. Case HB, Dickenson NE. Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using alpha-(32)P ATP. Bio Protoc. 2018;8(21). doi: 10.21769/BioProtoc.3074 30474049.

32. Demler HJ, Case HB, Morales Y, Bernard AR, Johnson SJ, Dickenson NE. Interfacial Amino Acids Support Spa47 Oligomerization and Shigella Type Three Secretion System Activation. Proteins. 2019. doi: 10.1002/prot.25754 31162724.

33. Niesel DW, Chambers CE, Stockman SL. Quantitation of HeLa cell monolayer invasion by Shigella and Salmonella species. J Clin Microbiol. 1985;22(6):897–902. 4066921.

34. Sansonetti PJ, Ryter A, Clerc P, Maurelli AT, Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infection and Immunity. 1986;51(2):461–9. 3510976

35. Parsot C, Ménard R, Gounon P, Sansonetti PJ. Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Molecular Microbiology. 1995;16(2):291–300. doi: 10.1111/j.1365-2958.1995.tb02301.x 7565091

36. Kenjale R, Wilson J, Zenk SF, Saurya S, Picking WL, Picking WD, et al. The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus. J Biol Chem. 2005;280(52):42929–37. doi: 10.1074/jbc.M508377200 16227202.

37. Bernard AR, Jessop TC, Kumar P, Dickenson NE. Deoxycholate-Enhanced Shigella Virulence Is Regulated by a Rare π-Helix in the Type Three Secretion System Tip Protein IpaD. Biochemistry. 2017;56(49):6503–14. doi: 10.1021/acs.biochem.7b00836 29134812.

38. Chatterjee R, Halder PK, Datta S. Identification and Molecular Characterization of YsaL (Ye3555): A Novel Negative Regulator of YsaN ATPase in Type Three Secretion System of Enteropathogenic Bacteria Yersinia enterocolitica. PLoS ONE. 2013;8(10). doi: 10.1371/journal.pone.0075028 24124464.

39. Pozidis C, Chalkiadaki A, Gomez-Serrano A, Stahlberg H, Brown I, Tampakaki AP, et al. Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization. The Journal of Biological Chemistry. 2003;278(28):25816–24. doi: 10.1074/jbc.M301903200 12734178.

40. Eichelberg K, Ginocchio CC, Galán JE. Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. Journal of Bacteriology. 1994;176(15):4501–10. doi: 10.1128/jb.176.15.4501-4510.1994 8045880.

41. Majewski DD, Worrall LJ, Hong C, Atkinson CE, Vuckovic M, Watanabe N, et al. Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry. Nature Communications. 2019;10. doi: 10.1038/s41467-019-08477-7 30733444

42. Stock D, Leslie AG, Walker JE. Molecular architecture of the rotary motor in ATP synthase. Science. 1999;286(5445):1700–5. doi: 10.1126/science.286.5445.1700 10576729.

43. Hu B, Lara-Tejero M, Kong Q, Galan JE, Liu J. In Situ Molecular Architecture of the Salmonella Type III Secretion Machine. Cell. 2017;168(6):1065–74 e10. doi: 10.1016/j.cell.2017.02.022 28283062.

44. Zhang Y, Lara-Tejero M, Bewersdorf J, Galan JE. Visualization and characterization of individual type III protein secretion machines in live bacteria. Proc Natl Acad Sci U S A. 2017;114(23):6098–103. doi: 10.1073/pnas.1705823114 28533372.

45. McMurry JL, Murphy JW, Gonzalez-Pedrajo B. The FliN-FliH interaction mediates localization of flagellar export ATPase FliI to the C ring complex. Biochemistry. 2006;45(39):11790–8. doi: 10.1021/bi0605890 17002279.


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