Modification of Pseudomonas aeruginosa Pa5196 Type IV Pilins at Multiple Sites with d -Ara f by a Novel GT-C Family Arabinosyltransferase, TfpW
10.1128/jb.01075-08
Crossref journal-article
American Society for Microbiology
Journal of Bacteriology (235)
Abstract

ABSTRACT Pseudomonas aeruginosa Pa5196 produces type IV pilins modified with unusual α1,5-linked d -arabinofuranose (α1,5- d -Ara f ) glycans, identical to those in the lipoarabinomannan and arabinogalactan cell wall polymers from Mycobacterium spp. In this work, we identify a second strain of P. aeruginosa , PA7, capable of expressing arabinosylated pilins and use a combination of site-directed mutagenesis, electrospray ionization mass spectrometry (MS), and electron transfer dissociation MS to identify the exact sites and extent of pilin modification in strain Pa5196. Unlike previously characterized type IV pilins that are glycosylated at a single position, those from strain Pa5196 were modified at multiple sites, with modifications of αβ-loop residues Thr64 and Thr66 being important for normal pilus assembly. Trisaccharides of α1,5- d -Ara f were the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues within the antiparallel beta sheet region of the pilin. TfpW was hypothesized to encode the pilin glycosyltransferase based on its genetic linkage to the pilin, weak similarity to membrane-bound GT-C family glycosyltransferases (which include the Mycobacterium arabinosyltransferases EmbA/B/C), and the presence of characteristic motifs. Loss of TfpW or mutation of key residues within the signature GT-C glycosyltransferase motif completely abrogated pilin glycosylation, confirming its involvement in this process. A Pa5196 pilA mutant complemented with other Pseudomonas pilins containing potential sites of modification expressed nonglycosylated pilins, showing that TfpW's pilin substrate specificity is restricted. TfpW is the prototype of a new type IV pilin posttranslational modification system and the first reported gram-negative member of the GT-C glycosyltransferase family.

Bibliography

Kus, J. V., Kelly, J., Tessier, L., Harvey, H., Cvitkovitch, D. G., & Burrows, L. L. (2008). Modification of Pseudomonas aeruginosa Pa5196 Type IV Pilins at Multiple Sites with d -Ara f by a Novel GT-C Family Arabinosyltransferase, TfpW. Journal of Bacteriology, 190(22), 7464–7478.

Authors 6
  1. Julianne V. Kus (first)
  2. John Kelly (additional)
  3. Luc Tessier (additional)
  4. Hanjeong Harvey (additional)
  5. Dennis G. Cvitkovitch (additional)
  6. Lori L. Burrows (additional)
References 64 Referenced 52
  1. Aas, F. E., A. Vik, J. Vedde, M. Koomey, and W. Egge-Jacobsen. 2007. Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure. Mol. Microbiol. 65 : 607-624. (10.1111/j.1365-2958.2007.05806.x) / Mol. Microbiol. (2007)
  2. Aas, F. E., W. Egge-Jacobsen, H. C. Winther-Larsen, C. Lovold, P. G. Hitchen, A. Dell, and M. Koomey. 2006. Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases. J. Biol. Chem. 281 : 27712-27723. (10.1074/jbc.M604324200) / J. Biol. Chem. (2006)
  3. Alaimo, C., I. Catrein, L. Morf, C. L. Marolda, N. Callewaert, M. A. Valvano, M. F. Feldman, and M. Aebi. 2006. Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides. EMBO J. 25 : 967-976. (10.1038/sj.emboj.7601024) / EMBO J. (2006)
  4. 10.1016/S0022-2836(05)80360-2
  5. 10.1128/JB.00938-08
  6. Benz, I., and M. A. Schmidt. 2002. Never say never again: protein glycosylation in pathogenic bacteria. Mol. Microbiol. 45 : 267-276. (10.1046/j.1365-2958.2002.03030.x) / Mol. Microbiol. (2002)
  7. Berg, S., D. Kaur, M. Jackson, and P. J. Brennan. 2007. The glycosyltransferases of Mycobacterium tuberculosis—roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates. Glycobiology 17 : 35R-56R. (10.1093/glycob/cwm010) / Glycobiology (2007)
  8. Blatch, G. L., and M. Lassle. 1999. The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 21 : 932-939. (10.1002/(SICI)1521-1878(199911)21:11<932::AID-BIES5>3.0.CO;2-N) / Bioessays (1999)
  9. Burrows, L. L. 2005. Weapons of mass retraction. Mol. Microbiol. 57 : 878-888. (10.1111/j.1365-2958.2005.04703.x) / Mol. Microbiol. (2005)
  10. Castric, P. 1995. pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin. Microbiology 141 : 1247-1254. (10.1099/13500872-141-5-1247) / Microbiology (1995)
  11. Chamot-Rooke, J., B. Rousseau, F. Lanternier, G. Mikaty, E. Mairey, C. Malosse, G. Bouchoux, V. Pelicic, L. Camoin, X. Nassif, and G. Dumenil. 2007. Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue. Proc. Natl. Acad. Sci. USA 104 : 14783-14788. (10.1073/pnas.0705335104) / Proc. Natl. Acad. Sci. USA (2007)
  12. Chenna, R., H. Sugawara, T. Koike, R. Lopez, T. J. Gibson, D. G. Higgins, and J. D. Thompson. 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31 : 3497-3500. (10.1093/nar/gkg500) / Nucleic Acids Res. (2003)
  13. 10.1128/JB.185.7.2374-2378.2003
  14. 10.1016/j.mimet.2005.06.001
  15. Coleman, F. T., S. Mueschenborn, G. Meluleni, C. Ray, V. J. Carey, S. O. Vargas, C. L. Cannon, F. M. Ausubel, and G. B. Pier. 2003. Hypersusceptibility of cystic fibrosis mice to chronic Pseudomonas aeruginosa oropharyngeal colonization and lung infection. Proc. Natl. Acad. Sci. USA 100 : 1949-1954. (10.1073/pnas.0437901100) / Proc. Natl. Acad. Sci. USA (2003)
  16. 10.1128/IAI.70.6.2837-2845.2002
  17. Coon, J. J., B. Ueberheide, J. E. Syka, D. D. Dryhurst, J. Ausio, J. Shabanowitz, and D. F. Hunt. 2005. Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 102 : 9463-9468. (10.1073/pnas.0503189102) / Proc. Natl. Acad. Sci. USA (2005)
  18. Coutinho, P. M., E. Deleury, G. J. Davies, and B. Henrissat. 2003. An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328 : 307-317. (10.1016/S0022-2836(03)00307-3) / J. Mol. Biol. (2003)
  19. Craig, L., M. E. Pique, and J. A. Tainer. 2004. Type IV pilus structure and bacterial pathogenicity. Nat. Rev. Microbiol. 2 : 363-378. (10.1038/nrmicro885) / Nat. Rev. Microbiol. (2004)
  20. Craig, L., R. K. Taylor, M. E. Pique, B. D. Adair, A. S. Arvai, M. Singh, S. J. Lloyd, D. S. Shin, E. D. Getzoff, M. Yeager, K. T. Forest, and J. A. Tainer. 2003. Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol. Cell 11 : 1139-1150. (10.1016/S1097-2765(03)00170-9) / Mol. Cell (2003)
  21. DiGiandomenico, A., M. J. Matewish, A. Bisaillon, J. R. Stehle, J. S. Lam, and P. Castric. 2002. Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity. Mol. Microbiol. 46 : 519-530. (10.1046/j.1365-2958.2002.03171.x) / Mol. Microbiol. (2002)
  22. 10.1128/iai.56.6.1641-1646.1988
  23. 10.1128/JB.01318-07
  24. Gallant, C. V., C. Daniels, J. M. Leung, A. S. Ghosh, K. D. Young, L. P. Kotra, and L. L. Burrows. 2005. Common beta-lactamases inhibit bacterial biofilm formation. Mol. Microbiol. 58 : 1012-1024. (10.1111/j.1365-2958.2005.04892.x) / Mol. Microbiol. (2005)
  25. Reference deleted.
  26. Giltner, C. L., E. J. van Schaik, G. F. Audette, D. Kao, R. S. Hodges, D. J. Hassett, and R. T. Irvin. 2006. The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces. Mol. Microbiol. 59 : 1083-1096. (10.1111/j.1365-2958.2005.05002.x) / Mol. Microbiol. (2006)
  27. Hahn, H. P. 1997. The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa—a review. Gene 192 : 99-108. (10.1016/S0378-1119(97)00116-9) / Gene (1997)
  28. Hegge, F. T., P. G. Hitchen, F. E. Aas, H. Kristiansen, C. Lovold, W. Egge-Jacobsen, M. Panico, W. Y. Leong, V. Bull, M. Virji, H. R. Morris, A. Dell, and M. Koomey. 2004. Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili. Proc. Natl. Acad. Sci. USA 101 : 10798-10803. (10.1073/pnas.0402397101) / Proc. Natl. Acad. Sci. USA (2004)
  29. Hoang, T. T., R. R. Karkhoff-Schweizer, A. J. Kutchma, and H. P. Schweizer. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212 : 77-86. (10.1016/S0378-1119(98)00130-9) / Gene (1998)
  30. Hogan, J. M., S. J. Pitteri, P. A. Chrisman, and S. A. McLuckey. 2005. Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation. J. Proteome Res. 4 : 628-632. (10.1021/pr049770q) / J. Proteome Res. (2005)
  31. Horton, R. M., Z. L. Cai, S. N. Ho, and L. R. Pease. 1990. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques 8 : 528-535. / BioTechniques (1990)
  32. Horzempa, J., J. E. Comer, S. A. Davis, and P. Castric. 2006. Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin. J. Biol. Chem. 281 : 1128-1136. (10.1074/jbc.M510975200) / J. Biol. Chem. (2006)
  33. Kikuchi, N., Y. D. Kwon, M. Gotoh, and H. Narimatsu. 2003. Comparison of glycosyltransferase families using the profile hidden Markov model. Biochem. Biophys. Res. Commun. 310 : 574-579. (10.1016/j.bbrc.2003.09.031) / Biochem. Biophys. Res. Commun. (2003)
  34. Kuo, C., N. Takahashi, A. F. Swanson, Y. Ozeki, and S. Hakomori. 1996. An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells. J. Clin. Investig. 98 : 2813-2818. (10.1172/JCI119109) / J. Clin. Investig. (1996)
  35. Kus, J. V., E. Tullis, D. G. Cvitkovitch, and L. L. Burrows. 2004. Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology 150 : 1315-1326. (10.1099/mic.0.26822-0) / Microbiology (2004)
  36. 10.1128/AEM.68.12.5956-5964.2002
  37. 10.1128/IAI.67.8.4084-4091.1999
  38. Liu, J., and A. Mushegian. 2003. Three monophyletic superfamilies account for the majority of the known glycosyltransferases. Protein Sci. 12 : 1418-1431. (10.1110/ps.0302103) / Protein Sci. (2003)
  39. Lu, H. M., S. T. Motley, and S. Lory. 1997. Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion. Mol. Microbiol. 25 : 247-259. (10.1046/j.1365-2958.1997.4561818.x) / Mol. Microbiol. (1997)
  40. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect. 2 : 1051-1060. (10.1016/S1286-4579(00)01259-4) / Microbes Infect. (2000)
  41. 10.1128/JB.181.2.656-661.1999
  42. Marceau, M., K. Forest, J. L. Beretti, J. Tainer, and X. Nassif. 1998. Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Mol. Microbiol. 27 : 705-715. (10.1046/j.1365-2958.1998.00706.x) / Mol. Microbiol. (1998)
  43. Mattick, J. S. 2002. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56 : 289-314. (10.1146/annurev.micro.56.012302.160938) / Annu. Rev. Microbiol. (2002)
  44. Murray, T. S., M. Egan, and B. I. Kazmierczak. 2007. Pseudomonas aeruginosa chronic colonization in cystic fibrosis patients. Curr. Opin. Pediatr. 19 : 83-88. (10.1097/MOP.0b013e3280123a5d) / Curr. Opin. Pediatr. (2007)
  45. O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30 : 295-304. (10.1046/j.1365-2958.1998.01062.x) / Mol. Microbiol. (1998)
  46. Power, P. M., and M. P. Jennings. 2003. The genetics of glycosylation in Gram-negative bacteria. FEMS Microbiol. Lett. 218 : 211-222. (10.1111/j.1574-6968.2003.tb11520.x) / FEMS Microbiol. Lett. (2003)
  47. Power, P. M., K. L. Seib, and M. P. Jennings. 2006. Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli. Biochem. Biophys. Res. Commun. 347 : 904-908. (10.1016/j.bbrc.2006.06.182) / Biochem. Biophys. Res. Commun. (2006)
  48. Romling, U., B. Fiedler, J. Bosshammer, D. Grothues, J. Greipel, H. von der Hardt, and B. Tummler. 1994. Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis. J. Infect. Dis. 170 : 1616-1621. (10.1093/infdis/170.6.1616) / J. Infect. Dis. (1994)
  49. Saiman, L., and A. Prince. 1993. Pseudomonas aeruginosa pili bind to asialoGM1 which is increased on the surface of cystic fibrosis epithelial cells. J. Clin. Investig. 92 : 1875-1880. (10.1172/JCI116779) / J. Clin. Investig. (1993)
  50. 10.1128/IAI.73.12.7922-7931.2005
  51. Spangenberg, C., R. Fislage, W. Sierralta, B. Tummler, and U. Romling. 1995. Comparison of type IV-pilin genes of Pseudomonas aeruginosa of various habitats has uncovered a novel unusual sequence. FEMS Microbiol. Lett. 125 : 265-273. (10.1111/j.1574-6968.1995.tb07367.x) / FEMS Microbiol. Lett. (1995)
  52. Spyropoulos, I. C., T. D. Liakopoulos, P. G. Bagos, and S. J. Hamodrakas. 2004. TMRPres2D: high quality visual representation of transmembrane protein models. Bioinformatics 20 : 3258-3260. (10.1093/bioinformatics/bth358) / Bioinformatics (2004)
  53. Stimson, E., M. Virji, S. Barker, M. Panico, I. Blench, J. Saunders, G. Payne, E. R. Moxon, A. Dell, and H. R. Morris. 1996. Discovery of a novel protein modification: alpha-glycerophosphate is a substituent of meningococcal pilin. Biochem. J. 316 : 29-33. (10.1042/bj3160029) / Biochem. J. (1996)
  54. Stimson, E., M. Virji, K. Makepeace, A. Dell, H. R. Morris, G. Payne, J. R. Saunders, M. P. Jennings, S. Barker, and M. Panico. 1995. Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose. Mol. Microbiol. 17 : 1201-1214. (10.1111/j.1365-2958.1995.mmi_17061201.x) / Mol. Microbiol. (1995)
  55. Strom, M. S., and S. Lory. 1993. Structure-function and biogenesis of the type IV pili. Annu. Rev. Microbiol. 47 : 565-596. (10.1146/annurev.mi.47.100193.003025) / Annu. Rev. Microbiol. (1993)
  56. 10.1128/jb.165.2.367-372.1986
  57. Syka, J. E., J. J. Coon, M. J. Schroeder, J. Shabanowitz, and D. F. Hunt. 2004. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. USA 101 : 9528-9533. (10.1073/pnas.0402700101) / Proc. Natl. Acad. Sci. USA (2004)
  58. Szymanski, C. M., and B. W. Wren. 2005. Protein glycosylation in bacterial mucosal pathogens. Nat. Rev. Microbiol. 3 : 225-237. (10.1038/nrmicro1100) / Nat. Rev. Microbiol. (2005)
  59. 10.1128/IAI.70.4.2242-2244.2002
  60. 10.1128/JB.01224-06
  61. Wall, D., and D. Kaiser. 1999. Type IV pili and cell motility. Mol. Microbiol. 32 : 1-10. (10.1046/j.1365-2958.1999.01339.x) / Mol. Microbiol. (1999)
  62. Wang, W., and B. A. Malcolm. 1999. Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis. BioTechniques 26 : 680-682. (10.2144/99264st03) / BioTechniques (1999)
  63. Wong, W. Y., A. P. Campbell, C. McInnes, B. D. Sykes, W. Paranchych, R. T. Irvin, and R. S. Hodges. 1995. Structure-function analysis of the adherence-binding domain on the pilin of Pseudomonas aeruginosa strains PAK and KB7. Biochemistry 34 : 12963-12972. (10.1021/bi00040a006) / Biochemistry (1995)
  64. Wuhrer, M., J. C. Stam, F. E. van de Geijn, C. A. Koeleman, C. T. Verrips, R. J. Dolhain, C. H. Hokke, and A. M. Deelder. 2007. Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum. Proteomics 7 : 4070-4081. (10.1002/pmic.200700289) / Proteomics (2007)
Dates
Type When
Created 16 years, 11 months ago (Sept. 19, 2008, 9:07 p.m.)
Deposited 4 years, 1 month ago (July 29, 2021, 1:43 p.m.)
Indexed 1 month ago (July 26, 2025, 5:07 a.m.)
Issued 16 years, 9 months ago (Nov. 15, 2008)
Published 16 years, 9 months ago (Nov. 15, 2008)
Published Print 16 years, 9 months ago (Nov. 15, 2008)
Funders 0

None

@article{Kus_2008, title={Modification of Pseudomonas aeruginosa Pa5196 Type IV Pilins at Multiple Sites with <scp>d</scp> -Ara f by a Novel GT-C Family Arabinosyltransferase, TfpW}, volume={190}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.01075-08}, DOI={10.1128/jb.01075-08}, number={22}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Kus, Julianne V. and Kelly, John and Tessier, Luc and Harvey, Hanjeong and Cvitkovitch, Dennis G. and Burrows, Lori L.}, year={2008}, month=nov, pages={7464–7478} }