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Understanding a Core Pilin of the Type IVa Pili of Acidithiobacillus thiooxidans, PilV

  • Araceli Hernandez-Sanchez (Geomicrobiologia, Metalurgia, Universidad Autonoma de San Luis Potosi) ;
  • Edgar D. Paez-Perez (Geomicrobiologia, Metalurgia, Universidad Autonoma de San Luis Potosi) ;
  • Elvia Alfaro-Saldana (Geomicrobiologia, Metalurgia, Universidad Autonoma de San Luis Potosi) ;
  • Vanesa Olivares-Illana (Laboratorio de Interacciones Biomoleculares y Cancer. Instituto de Fisica, Universidad Autonoma de San Luis Potosi) ;
  • J. Viridiana Garcia-Meza (Geomicrobiologia, Metalurgia, Universidad Autonoma de San Luis Potosi)
  • Received : 2023.10.25
  • Accepted : 2023.12.29
  • Published : 2024.03.28

Abstract

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

Keywords

Acknowledgement

This work was supported by the National Council of Humanities Science and Technology (CONAHCyT-CB2017-2018 Project A1-S-11505). EDPP acknowledges the support of this research by a postdoctoral scholarship from CONAHCyT (I1200/311/2023). Thanks are expressed to Fernando Rangel for his experimental support and to Alfredo Mendez for suggestions on methodologies for heterologous cloning of PilV. Finally, to Margaret Schroeder for her valuable comments.

References

  1. Pelicic V. 2008. Type IV pili: e pluribus unum?? Mol. Microbiol. 68: 827-837. https://doi.org/10.1111/j.1365-2958.2008.06197.x
  2. Gerven N, Waksman G, Remaut H. 2011. Pili and flagella: biology, structure, and biotechnological applications. Prog. Mol. Biol. Transl. Sci. 103: 21-72. https://doi.org/10.1016/B978-0-12-415906-8.00005-4
  3. Conrad JC. 2012. Physics of bacterial near-surface motility using flagella and type IV pili: implications for biofilm formation. Res. Microbiol. 163: 619-629. https://doi.org/10.1016/j.resmic.2012.10.016
  4. Craig L, Forest KT, Maier B. 2019. Type IV pili: dynamics, biophysics and functional consequences. Nat. Rev. Microbiol. 17: 429-440. https://doi.org/10.1038/s41579-019-0195-4
  5. Craig L, Li J. 2008. Type IV pili: paradoxes in form and function. Curr. Opin. Struct. Biol. 18: 267-277. https://doi.org/10.1016/j.sbi.2007.12.009
  6. Jacobsen T, Bardiaux B, Francetic O, Izadi-Pruneyre N, Nilges M. 2020. Structure and function of minor pilins of type IV pili. Med. Microbiol. Immunol. 209: 301-308. https://doi.org/10.1007/s00430-019-00642-5
  7. Helaine S, Dyer DH, Nassif X, Pelicic V, Forest KT. 2007. 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili. Proc. Natl. Acad. Sci. USA 104: 15888-15893. https://doi.org/10.1073/pnas.0707581104
  8. Maier B, Potter L, So M, Seifert HS, Sheetz MP. 2002. Single pilus motor forces exceed 100 pN. Proc. Natl. Acad. Sci. USA 99: 16012-16017. https://doi.org/10.1073/pnas.242523299
  9. Berry JL, Pelicic V. 2015. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol. Rev. 39: 134-154. https://doi.org/10.1093/femsre/fuu001
  10. Singhi D, Srivastava P. 2020. Role of bacterial cytoskeleton and other apparatuses in cell communication. Front. Mol. Biosci. 7: 158.
  11. Li Y, Huang S, Zhang X, Huang T, Li H. 2013. Cloning, expression, and functional analysis of molecular motor pilT and pilU genes of type IV pili in Acidithiobacillus ferrooxidans. Appl. Microbiol. Biotechnol. 97: 1251-1257. https://doi.org/10.1007/s00253-012-4271-1
  12. Tu B, Wang F, Li J, Sha J, Lu X, Han X. 2013. Analysis of genes and proteins in Acidithiobacillus ferrooxidans during growth and attachment on pyrite under different conditions. Geomicrobiol. J. 30: 255-267. https://doi.org/10.1080/01490451.2012.668608
  13. Li Y, Li H. 2014. Type IV pili of Acidithiobacillus ferrooxidans can transfer electrons from extracellular electron donors. J. Basic Microbiol. 54: 226-231. https://doi.org/10.1002/jobm.201200300
  14. Malvankar NS, Lovley DR. 2014. Microbial nanowires for bioenergy applications. Curr. Opin. Biotechnol. 27: 88-95. https://doi.org/10.1016/j.copbio.2013.12.003
  15. Piepenbrink KH. 2019. DNA uptake by type IV filaments. Front. Mol. Biosci. 6: 1.
  16. Lukaszczyk M, Pradhan B, Remaut H. 2019. The biosynthesis and structures of bacterial pili, pp. 369-413. In Kuhn A (ed.), Bacterial Cell Walls and Membranes. Springer, Cham. doi:10.1007/978-3-030-18768-2_12.
  17. Raynaud C. 2021. Understanding type IV pili mediated adhesion in Streptococcus sanguinis. PhD Dissertation. Imperial College, London. doi:10.25560/92922.
  18. Valdes J, Pedroso I, Quatrini R, Dodson RJ, Tettelin H, Blake R, Eisen JA, Holmes DS. 2008. Acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications. BMC Genomics 9: 597.
  19. Diaz- Beneventi M, Castro M, Copaja-Castillo S, Guiliani-Guerin N. 2018. Biofilm formation by the acidophile bacterium Acidithiobacillus thiooxidans Involves c-di-GMP Pathway and Pel exopolysaccharide. Genes 9: 113. doi:10.3390/genes9020113.
  20. Alfaro-Saldana E, Hernandez-Sanchez A, Araceli Patron-Soberano OA, Astello-Garcia M, Mendez-Cabanas JA, Garcia-Meza JV. 2019. Sequence analysis and confirmation of the type IV pili-associated proteins PilY1, PilW and PilV in Acidithiobacillus thiooxidans. PLoS One 14: e.0199854.
  21. Giltner CL, Habash M, Burrows LL. 2010. Pseudomonas aeruginosa minor pilins are incorporated into type iv pili. J. Mol. Biol. 398: 444-461. https://doi.org/10.1016/j.jmb.2010.03.028
  22. Hospenthal MK, Costa TRD, Waksman G. 2017. A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat. Rev. Microbiol. 15: 365-379. https://doi.org/10.1038/nrmicro.2017.40
  23. Bardy SL, Ng SYM, Jarrell KF. 2003. Prokaryotic motility structures. Microbiol. 149: 295-304. https://doi.org/10.1099/mic.0.25948-0
  24. Linhartova M, Skotnicova P, Hakkila K, Tichy M, Komenda J, Knoppova J, et al. 2021. Mutations suppressing the lack of prepilin peptidase provide insights into the maturation of the major pilin protein in cyanobacteria. Front. Microbiol. 12: 756912.
  25. Garcia-Meza JV, Fernandez JJ, Lara RH, Gonzalez I. 2013. Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans. Appl. Microbiol. Biotechnol. 97: 6065-6075. https://doi.org/10.1007/s00253-012-4420-6
  26. Yang L, Zhao D, Yang J, Wang W, Chen P, Zhang S, Yan L. 2019. Acidithiobacillus thiooxidans and its potential application. Appl. Microbiol. Biotechnol. 103: 7819-7833. https://doi.org/10.1007/s00253-019-10098-5
  27. Alm RA, Mattick JS. 1997. Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192: 89-98. https://doi.org/10.1016/S0378-1119(96)00805-0
  28. Nguyen Y, Sugiman-Marangos S, Harvey H, Bell SD, Charlton CL, Junop MS, Burrows LL. 2015. Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin. J. Biol. Chem. 290: 601-611. https://doi.org/10.1074/jbc.M114.616904
  29. McCallum M, Burrows LL, Howell PL. 2019. The dynamic structures of the type IV pilus. Microbiol. Spectr. 7: 10-1128. https://doi.org/10.1128/microbiolspec.PSIB-0006-2018
  30. Craig L, Pique ME, Tainer JA. 2004. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2: 363-378. https://doi.org/10.1038/nrmicro885
  31. Tang D, Gao Q, Zhao Y, Li Y, Chen P, Zhou J, et al. 2018. Mg2+ reduces biofilm quantity in Acidithiobacillus ferrooxidans through inhibiting Type IV pili formation. FEMS Microbiol. Lett. 365: fnx266.
  32. Paez-Perez ED, Hernandez-Sanchez A, Alfaro-Saldana E, Garcia-Meza JV. 2023. Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows. Extremophiles 27: 31.
  33. Feng S, Li K, Huang Z, Tong Y, Yang H. 2019. Specific mechanism of Acidithiobacillus caldus extracellular polymeric substances in the bioleaching of copper-bearing sulfide ore. PLoS One 14: e0213945.
  34. Boase K, Gonzalez C, Vergara E, Neira G, Holmes D, Watkin E. 2022. Prediction and inferred evolution of acid tolerance genes in the biotechnologically important Acidihalobacter genus. Front. Microbiol. 13: 848410.
  35. Gonzalez-Rosales C, Vergara E, Dopson M, Valdes JH, Holmes DS. 2021. Integrative genomics sheds light on evolutionary forces shaping the Acidithiobacillia class acidophilic lifestyle. Front. Microbiol. 12: 822229.
  36. Wang F, Baquero DP, Su Z, Beltran LC, Prangishvili D, Krupovic M, et al. 2020. The structures of two archaeal type IV pili illuminate evolutionary relationships. Nat. Commun. 11: 3424.
  37. Flores-Rios R, Moya-Beltran A, Pareja-Barrueto C, Arenas-Salinas M, Valenzuela S, Orellana O, et al. 2019. The type IV secretion system of ICEAfe1: formation of a conjugative pilus in Acidithiobacillus ferrooxidans. Front. Microbiol. 10: 30.
  38. LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Nat. Biotechnol. 11: 187-193. https://doi.org/10.1038/nbt0293-187
  39. Liu ZQ, Yang PC. 2012. Construction of pET-32 α (+) vector for protein expression and purification. N. Am. J. Med. Sci. 4: 651-655. https://doi.org/10.4103/1947-2714.104318
  40. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  41. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589. https://doi.org/10.1038/s41586-021-03819-2
  42. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. 2004. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25: 1605-1612. https://doi.org/10.1002/jcc.20084
  43. Xue B, Dunbrack RL, Williams RW, Dunker AK, Uversky VN. 2010. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim. Biophys. Acta - Proteins Proteom. 1804: 996-1010. https://doi.org/10.1016/j.bbapap.2010.01.011
  44. McGuffin LJ, Bryson K, Jones DT. 2000. The PSIPRED protein structure prediction server. Bioinformatics 16: 404-405. https://doi.org/10.1093/bioinformatics/16.4.404
  45. Hallgren J, Tsirigos KD, Damgaard Pedersen M, Juan J, Armenteros A, Marcatili P, et al. 2022. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. bioRxiv. 2022.04.08.487609 doi:10.1101/2022.04.08.487609.
  46. Hebditch M, Warwicker J. 2019. Web-based display of protein surface and pH-dependent properties for assessing the developability of biotherapeutics. Sci. Rep. 9: 1969.
  47. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. 2005. Protein identification and analysis tools on the ExPASy server, pp. 571-607. In Walker JM (ed.), The Proteomics Protocols Handbook. Springer Berlin. doi:10.1385/1-59259-890-0:571.
  48. Fink AL, Calciano LJ, Goto Y, Kurotsu T, Palleros DR. 1994. Classification of acid denaturation of proteins: intermediates and unfolded states. Biochemistry 33: 12504-12511. https://doi.org/10.1021/bi00207a018
  49. Almutairi GO, Malik A, Alonazi M, Khan JM, Alhomida AS, Khan MS, Alenad AM, Altwaijry N, Alafaleq NO. 2022. Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease. Int. J. Biol. Macromol. 209: 984-990. https://doi.org/10.1016/j.ijbiomac.2022.04.077
  50. Jazaj D, Ghadami SA, Bemporad F, Chiti F. 2019. Probing conformational changes of monomeric transthyretin with second derivative fluorescence. Sci. Rep. 9: 10988.
  51. Muhlmann M, Forsten E, Noack S, Buchs J. 2017. Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb. Cell Fact. 16: 220.
  52. Bartolo-Aguilar Y, Chavez-Cabrera C, Flores-Cotera LB, Badillo-Corona JA, Oliver-Salvador C, Marsch R. 2022. The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells. J. Genet. Eng. Biotechnol. 20: 173.
  53. Shanmugasundarasamy T, Karaiyagowder Govindarajan D, Kandaswamy K. 2022. A review on pilus assembly mechanisms in Gram-positive and Gram-negative bacteria. Cell Surf. 8: 100077.
  54. Sahin C, Reid DJ, Marty MT, Landreh M. 2020. Scratching the surface: native mass spectrometry of peripheral membrane protein complexes. Biochem. Soc. Trans. 48: 547-558. https://doi.org/10.1042/BST20190787
  55. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, et al. 2017. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 45(D): 200-203. https://doi.org/10.1093/nar/gkw1129
  56. Holmes DE, Dang Y, Walker DJF, Lovley DR. 2016. The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microb. Genom. 2: e000072.
  57. Nguyen Y, Boulton S, McNicholl ET, Akimoto M, Harvey H, Aidoo F, et al. 2018. A highly dynamic loop of the Pseudomonas aeruginosa PA14 type IV pilin is essential for pilus assembly. ACS Infect. Dis. 4: 936-943. https://doi.org/10.1021/acsinfecdis.7b00229
  58. Giltner CL, Nguyen Y, Burrows LL. 2012. Type IV pilin proteins: versatile molecular modules. Microbiol. Mol. Biol. Rev. 76: 740-772. https://doi.org/10.1128/MMBR.00035-12
  59. Harvey H, Habash M, Aidoo F, Burrows LL. 2009. Single-residue changes in the c-terminal disulfide-bonded loop of the Pseudomonas aeruginosa type IV pilin influence pilus assembly and twitching motility. J. Bacteriol. 191: 6513-6524. https://doi.org/10.1128/JB.00943-09
  60. Lee KK, Sheth HB, Wong WY, Sherburne R, Paranchych W, Hodges RS, et al. 1994. The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit. Mol. Microbiol. 11: 705-713. https://doi.org/10.1111/j.1365-2958.1994.tb00348.x
  61. Li J, Egelman EH, Craig L. 2012. Structure of the Vibrio cholerae type IVb pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus. J. Mol. Biol. 418: 47-64. https://doi.org/10.1016/j.jmb.2012.02.017
  62. Zhang RM, Snyder GH. 1989. Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids. J. Biol. Chem. 264: 18472-18479. https://doi.org/10.1016/S0021-9258(18)51490-3
  63. Hartung S, Arvai AS, Wood T, Kolappan S, Shin DS, Craig L, Tainer JA. 2011. Ultrahigh resolution and full-length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential. J. Biol. Chem. 286: 44254-44265. https://doi.org/10.1074/jbc.M111.297242
  64. Rojas-Chapana JA, Tributsch H. 2000. Bio-leaching of pyrite accelerated by cysteine. Process Biochem. 35: 815-824. https://doi.org/10.1016/S0032-9592(99)00142-9
  65. Ye J-P, Wang S-Q, Zhang P-Y, Nabi M, Tao X, Zhang H-B, et al. 2020. L-cysteine addition enhances microbial surface oxidation of coal inorganic sulfur: complexation of cysteine and pyrite, inhibition of jarosite formation, environmental effects. Environ. Res. 187: 109705.
  66. Luzar A. 2000. Resolving the hydrogen bond dynamics conundrum. J. Chem. Phys. 113: 10663-10675. https://doi.org/10.1063/1.1320826
  67. Zahn S, Wendler K, Delle Site L, Kirchner B. 2011. Depolarization of water in protic ionic liquids. Phys. Chem. Chem. Phys. 13: 15083.
  68. Bezrodnyi V, Shavykin O, Mikhtaniuk S, Neelov IM, Markelov DA. 2020. Molecular dynamics and spin-lattice NMR relaxation α-and ε-polylysine. Appl. Magn. Reson. 51: 1669-1679. https://doi.org/10.1007/s00723-020-01260-8
  69. Zhou HX, Pang X. 2018. Electrostatic interactions in protein structure, folding, binding, and condensation. Chem. Rev. 118: 1691-1741. https://doi.org/10.1021/acs.chemrev.7b00305
  70. Watanabe H, Yoshida C, Ooishi A, Nakai Y, Ueda M, Isobe Y, et al. 2019. Histidine-mediated intramolecular electrostatic repulsion for controlling pH-dependent protein-protein interaction. ACS Chem. Biol. 14: 2729-2736. https://doi.org/10.1021/acschembio.9b00652
  71. Niemann T, Stange P, Strate A, Ludwig R. 2019. When hydrogen bonding overcomes Coulomb repulsion: from kinetic to thermodynamic stability of cationic dimers. Phys. Chem. Chem. Phys. 21: 8215-8220. https://doi.org/10.1039/C8CP06417B