PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

English version

Autoři: Jennifer L. Chlebek ^aff001; Hannah Q. Hughes ^aff001; Aleksandra S. Ratkiewicz ^aff002; Rasman Rayyan ^aff002; Joseph Che-Yen Wang ^aff003; Brittany E. Herrin ^aff001; Triana N. Dalia ^aff001; Nicolas Biais ^aff002; Ankur B. Dalia ^aff001
Působiště autorů: Department of Biology, Indiana University, Bloomington, Indiana, United States of America ^aff001; Biology Department and Graduate Center, City University of New York, Brooklyn, New York, United States of America ^aff002; Electron Microscopy Center, Indiana University, Bloomington, Indiana, United States of America ^aff003
Vyšlo v časopise: PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008448
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008448

Souhrn

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.

Klíčová slova:

Adenosine triphosphatase – Fluorescence imaging – Glycerol – Hyperexpression techniques – Mutant strains – Phenotypes – Pili and fimbriae – Vibrio cholerae

Zdroje

1. Burrows LL. Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu Rev Microbiol. 2012;66:493–520. Epub 2012/07/04. doi: 10.1146/annurev-micro-092611-150055 22746331.

2. Maier B, Wong GCL. How Bacteria Use Type IV Pili Machinery on Surfaces. Trends Microbiol. 2015;23(12):775–88. doi: 10.1016/j.tim.2015.09.002 26497940.

3. Hospenthal MK, Costa TRD, Waksman G. A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat Rev Microbiol. 2017;15(6):365–79. Epub 2017/05/13. doi: 10.1038/nrmicro.2017.40 28496159.

4. Proft T, Baker EN. Pili in Gram-negative and Gram-positive bacteria—structure, assembly and their role in disease. Cell Mol Life Sci. 2009;66(4):613–35. Epub 2008/10/28. doi: 10.1007/s00018-008-8477-4 18953686.

5. Craig L, Forest KT, Maier B. Type IV pili: dynamics, biophysics and functional consequences. Nat Rev Microbiol. 2019. Epub 2019/04/17. doi: 10.1038/s41579-019-0195-4 30988511.

6. Craig L, Pique ME, Tainer JA. Type IV pilus structure and bacterial pathogenicity. Nat Rev Microbiol. 2004;2(5):363–78. doi: 10.1038/nrmicro885 15100690.

7. McCallum M, Tammam S, Khan A, Burrows LL, Howell PL. The molecular mechanism of the type IVa pilus motors. Nat Commun. 2017;8:15091. doi: 10.1038/ncomms15091 28474682; PubMed Central PMCID: PMC5424180.

8. Bischof LF, Friedrich C, Harms A, Sogaard-Andersen L, van der Does C. The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM. J Biol Chem. 2016;291(13):6946–57. doi: 10.1074/jbc.M115.701284 26851283; PubMed Central PMCID: PMC4807279.

9. Chang YW, Rettberg LA, Treuner-Lange A, Iwasa J, Sogaard-Andersen L, Jensen GJ. Architecture of the type IVa pilus machine. Science. 2016;351(6278):aad2001. doi: 10.1126/science.aad2001 26965631; PubMed Central PMCID: PMC5929464.

10. Chiang P, Habash M, Burrows LL. Disparate subcellular localization patterns of Pseudomonas aeruginosa Type IV pilus ATPases involved in twitching motility. J Bacteriol. 2005;187(3):829–39. doi: 10.1128/JB.187.3.829-839.2005 PubMed Central PMCID: PMC545728. 15659660

11. Craig L, Volkmann N, Arvai AS, Pique ME, Yeager M, Egelman EH, et al. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol Cell. 2006;23(5):651–62. doi: 10.1016/j.molcel.2006.07.004 16949362.

12. Jakovljevic V, Leonardy S, Hoppert M, Sogaard-Andersen L. PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus. J Bacteriol. 2008;190(7):2411–21. doi: 10.1128/JB.01793-07 18223089; PubMed Central PMCID: PMC2293208.

13. Whitchurch CB, Mattick JS. Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa. Mol Microbiol. 1994;13(6):1079–91. Epub 1994/09/01. doi: 10.1111/j.1365-2958.1994.tb00499.x 7854122.

14. Blair KM, Turner L, Winkelman JT, Berg HC, Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science. 2008;320(5883):1636–8. Epub 2008/06/21. doi: 10.1126/science.1157877 18566286.

15. Ellison CK, Kan J, Dillard RS, Kysela DT, Ducret A, Berne C, et al. Obstruction of pilus retraction stimulates bacterial surface sensing. Science. 2017;358(6362):535–8. Epub 2017/10/28. doi: 10.1126/science.aan5706 29074778; PubMed Central PMCID: PMC5805138.

16. Ellison CK, Dalia TN, Dalia AB, Brun YV. Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules. Nat Protoc. 2019. doi: 10.1038/s41596-019-0162-6 31028374.

17. Adams DW, Stutzmann S, Stoudmann C, Blokesch M. DNA-uptake pili of Vibrio cholerae are required for chitin colonization and capable of kin recognition via sequence-specific self-interaction. Nat Microbiol. 2019. doi: 10.1038/s41564-019-0479-5 31182799.

18. Ellison CK, Dalia TN, Vidal Ceballos A, Wang JC, Biais N, Brun YV, et al. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nat Microbiol. 2018;3(7):773–80. Epub 2018/06/13. doi: 10.1038/s41564-018-0174-y 29891864.

19. Seitz P, Blokesch M. DNA-uptake machinery of naturally competent Vibrio cholerae. Proc Natl Acad Sci U S A. 2013;110(44):17987–92. Epub 2013/10/16. doi: 10.1073/pnas.1315647110 24127573; PubMed Central PMCID: PMC3816411.

20. Bieber D, Ramer SW, Wu CY, Murray WJ, Tobe T, Fernandez R, et al. Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science. 1998;280(5372):2114–8. doi: 10.1126/science.280.5372.2114 9641917.

21. Chamot-Rooke J, Mikaty G, Malosse C, Soyer M, Dumont A, Gault J, et al. Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination. Science. 2011;331(6018):778–82. doi: 10.1126/science.1200729 21311024.

22. Helaine S, Carbonnelle E, Prouvensier L, Beretti JL, Nassif X, Pelicic V. PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol Microbiol. 2005;55(1):65–77. doi: 10.1111/j.1365-2958.2004.04372.x 15612917.

23. Dietrich M, Mollenkopf H, So M, Friedrich A. Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11. FEMS Microbiol Lett. 2009;296(2):248–56. doi: 10.1111/j.1574-6968.2009.01647.x 19486161; PubMed Central PMCID: PMC4428587.

24. Merz AJ, So M, Sheetz MP. Pilus retraction powers bacterial twitching motility. Nature. 2000;407(6800):98–102. doi: 10.1038/35024105 10993081.

25. Pujol C, Eugene E, Marceau M, Nassif X. The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion. Proc Natl Acad Sci U S A. 1999;96(7):4017–22. doi: 10.1073/pnas.96.7.4017 10097155; PubMed Central PMCID: PMC22412.

26. Hockenberry AM, Hutchens DM, Agellon A, So M. Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior. MBio. 2016;7(6). doi: 10.1128/mBio.01994-16 27923924; PubMed Central PMCID: PMC5142622.

27. Wolfgang M, Lauer P, Park HS, Brossay L, Hebert J, Koomey M. PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol. 1998;29(1):321–30. doi: 10.1046/j.1365-2958.1998.00935.x 9701824.

28. Leighton TL, Dayalani N, Sampaleanu LM, Howell PL, Burrows LL. Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations. J Bacteriol. 2015;197(13):2229–38. doi: 10.1128/JB.00220-15 25917913; PubMed Central PMCID: PMC4455263.

29. Aukema KG, Kron EM, Herdendorf TJ, Forest KT. Functional dissection of a conserved motif within the pilus retraction protein PilT. J Bacteriol. 2005;187(2):611–8. doi: 10.1128/JB.187.2.611-618.2005 15629932; PubMed Central PMCID: PMC543540.

30. Hanson PI, Whiteheart SW. AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol. 2005;6(7):519–29. doi: 10.1038/nrm1684 16072036.

31. McCallum M, Benlekbir S, Nguyen S, Tammam S, Rubinstein JL, Burrows LL, et al. Multiple conformations facilitate PilT function in the type IV pilus. bioRxiv. 2019. https://doi.org/10.1101/672212.

32. Tala L, Fineberg A, Kukura P, Persat A. Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements. Nat Microbiol. 2019. Epub 2019/02/26. doi: 10.1038/s41564-019-0378-9 30804544.

33. Biais N, Higashi D, So M, Ladoux B. Techniques to measure pilus retraction forces. Methods Mol Biol. 2012;799:197–216. doi: 10.1007/978-1-61779-346-2_13 21993648; PubMed Central PMCID: PMC5160128.

34. Zollner R, Cronenberg T, Maier B. Motor properties of PilT-independent type 4 pilus retraction in gonococci. J Bacteriol. 2019. doi: 10.1128/JB.00778-18 30692169

35. Georgiadou M, Castagnini M, Karimova G, Ladant D, Pelicic V. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly. Mol Microbiol. 2012;84(5):857–73. doi: 10.1111/j.1365-2958.2012.08062.x 22486968.

36. Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A. 1998;95(10):5752–6. Epub 1998/05/20. doi: 10.1073/pnas.95.10.5752 9576956; PubMed Central PMCID: PMC20451.

37. McCallum M, Tammam S, Little DJ, Robinson H, Koo J, Shah M, et al. PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM. J Biol Chem. 2016;291(21):11003–15. doi: 10.1074/jbc.M116.718353 27022027; PubMed Central PMCID: PMC4900251.

38. Forest KT, Satyshur KA, Worzalla GA, Hansen JK, Herdendorf TJ. The pilus-retraction protein PilT: ultrastructure of the biological assembly. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 5):978–82. Epub 2004/04/23. doi: 10.1107/S0907444904006055 15103158.

39. Jones CJ, Utada A, Davis KR, Thongsomboon W, Zamorano Sanchez D, Banakar V, et al. C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae. PLoS Pathog. 2015;11(10):e1005068. doi: 10.1371/journal.ppat.1005068 26505896; PubMed Central PMCID: PMC4624765.

40. Takhar HK, Kemp K, Kim M, Howell PL, Burrows LL. The platform protein is essential for type IV pilus biogenesis. J Biol Chem. 2013;288(14):9721–8. doi: 10.1074/jbc.M113.453506 23413032; PubMed Central PMCID: PMC3617274.

41. Adams DW, Pereira JM, Stoudmann C, Stutzmann S, Blokesch M. The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase. PLoS Genet. 2019;15(9):e1008393. doi: 10.1371/journal.pgen.1008393 31525185.

42. Chiang P, Sampaleanu LM, Ayers M, Pahuta M, Howell PL, Burrows LL. Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU. Microbiology. 2008;154(Pt 1):114–26. doi: 10.1099/mic.0.2007/011320-0 18174131.

43. Ellison CK, Kan J, Chlebek JL, Hummels KR, Panis G, Viollier PH, et al. A bifunctional ATPase drives tad pilus extension and retraction. bioRxiv. 2019:616128. doi: 10.1101/616128

44. Juni E, Janik A. Transformation of Acinetobacter calco-aceticus (Bacterium anitratum). J Bacteriol. 1969;98(1):281–8. 5781579; PubMed Central PMCID: PMC249934.

45. Dalia AB. Natural Cotransformation and Multiplex Genome Editing by Natural Transformation (MuGENT) of Vibrio cholerae. Methods Mol Biol. 2018;1839:53–64. Epub 2018/07/27. doi: 10.1007/978-1-4939-8685-9_6 30047054.

46. Dalia AB, McDonough E, Camilli A. Multiplex genome editing by natural transformation. Proc Natl Acad Sci U S A. 2014;111(24):8937–42. Epub 2014/06/04. doi: 10.1073/pnas.1406478111 24889608; PubMed Central PMCID: PMC4066482.

47. Dalia TN, Yoon SH, Galli E, Barre FX, Waters CM, Dalia AB. Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases. Nucleic Acids Res. 2017;45(12):7527–37. Epub 2017/06/03. doi: 10.1093/nar/gkx496 28575400; PubMed Central PMCID: PMC5499599.

48. Dalia AB, Lazinski DW, Camilli A. Characterization of undermethylated sites in Vibrio cholerae. J Bacteriol. 2013;195(10):2389–99. Epub 2013/03/19. doi: 10.1128/JB.02112-12 23504020; PubMed Central PMCID: PMC3650525.

49. Zhu J, Miller MB, Vance RE, Dziejman M, Bassler BL, Mekalanos JJ. Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A. 2002;99(5):3129–34. doi: 10.1073/pnas.052694299 11854465; PubMed Central PMCID: PMC122484.

50. Lo Scrudato M, Blokesch M. The regulatory network of natural competence and transformation of Vibrio cholerae. PLoS Genet. 2012;8(6):e1002778. doi: 10.1371/journal.pgen.1002778 22737089; PubMed Central PMCID: PMC3380833.

51. Lo Scrudato M, Blokesch M. A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable. Nucleic Acids Res. 2013;41(6):3644–58. doi: 10.1093/nar/gkt041 23382174; PubMed Central PMCID: PMC3616704.

52. Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK. Chitin induces natural competence in Vibrio cholerae. Science. 2005;310(5755):1824–7. doi: 10.1126/science.1120096 16357262.

53. Dalia AB, Lazinski DW, Camilli A. Identification of a membrane-bound transcriptional regulator that links chitin and natural competence in Vibrio cholerae. MBio. 2014;5(1):e01028–13. Epub 2014/01/30. doi: 10.1128/mBio.01028-13 24473132; PubMed Central PMCID: PMC3903286.

54. Nero TM, Dalia TN, Wang JC, Kysela DT, Bochman ML, Dalia AB. ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species. Nucleic Acids Res. 2018;46(12):6099–111. Epub 2018/05/04. doi: 10.1093/nar/gky343 29722872; PubMed Central PMCID: PMC6158740.

55. Ludtke SJ. Single-Particle Refinement and Variability Analysis in EMAN2.1. Methods Enzymol. 2016;579:159–89. Epub 2016/08/31. doi: 10.1016/bs.mie.2016.05.001 PubMed Central PMCID: PMC5101015. 27572727

56. Scheres SH. Semi-automated selection of cryo-EM particles in RELION-1.3. J Struct Biol. 2015;189(2):114–22. Epub 2014/12/09. doi: 10.1016/j.jsb.2014.11.010 25486611; PubMed Central PMCID: PMC4318617.

57. Satyshur KA, Worzalla GA, Meyer LS, Heiniger EK, Aukema KG, Misic AM, et al. Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure. 2007;15(3):363–76. doi: 10.1016/j.str.2007.01.018 17355871; PubMed Central PMCID: PMC1978094.

58. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. Epub 2004/07/21. doi: 10.1002/jcc.20084 15264254.