1887

Abstract

The heat-resistant agglutinin 1 (Hra1) is an integral outer membrane protein found in strains of that are exceptional colonizers. Hra1 from enteroaggregative strain 042 is sufficient to confer adherence to human epithelial cells and to cause bacterial autoaggregation. Hra1 is closely related to the Tia invasin, which also confers adherence, but not autoaggregation. Here, we have demonstrated that Hra1 mediates autoaggregation by self-association and we hypothesize that at least some surface-exposed amino acid sequences that are present in Hra1, but absent in Tia, represent autoaggregation motifs. We inserted FLAG tags along the length of Hra1 and used immune-dot blots to verify that four -predicted outer loops were indeed surface exposed. In Hra1 we swapped nine candidate motifs in three of these loops, ranging from one to ten amino acids in length, to the corresponding sequences in Tia. Three of the motifs were required for Hra1-mediated autoaggregation. The database was searched for other surface proteins containing these motifs; the GGXWRDDXK motif was also present in a surface-exposed region of Rck, a serotype Typhimurium complement resistance protein. Cloning and site-specific mutagenesis demonstrated that Rck can confer weak, GGXWRDDXK-dependent autoaggregation by self-association. Hra1 and Rck appear to form heterologous associations and GGXWRDDXK is required on both molecules for Hra1–Rck association. However, a GGYWRDDLKE peptide was not sufficient to interfere with Hra1-mediated autoaggregation. In the present study, three autoaggregation motifs in an integral outer membrane protein have been identified and it was demonstrated that at least one of them works in the context of a different cell surface.

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2016-07-01
2024-03-28
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References

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [View Article][PubMed]
    [Google Scholar]
  2. Barondess J. J., Beckwith J. 1990; A bacterial virulence determinant encoded by lysogenic coliphage lambda. Nature 346:871–874 [View Article][PubMed]
    [Google Scholar]
  3. Bhargava S., Johnson B. B., Hwang J., Harris T. A., George A. S., Muir A., Dorff J., Okeke I. N. 2009; Heat-resistant agglutinin 1 is an accessory enteroaggregative Escherichia coli colonization factor. J Bacteriol 191:4934–4942 [View Article][PubMed]
    [Google Scholar]
  4. Chart H., Spencer J., Smith H. R., Rowe B. 1997; Identification of entero-aggregative Escherichia coli based on surface properties. J Appl Microbiol 83:712–717[PubMed] [CrossRef]
    [Google Scholar]
  5. Chaudhuri R. R., Sebaihia M., Hobman J. L., Webber M. A., Leyton D. L., Goldberg M. D., Cunningham A. F., Scott-Tucker A., Ferguson P. R. et al. 2010; Complete genome sequence and comparative metabolic profiling of the prototypical enteroaggregative Escherichia coli strain 042. PLoS One 5:e8801 [View Article][PubMed]
    [Google Scholar]
  6. Chenna R., Sugawara H., Koike T., Lopez R., Gibson T. J., Higgins D. G., Thompson J. D. 2003; Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500 [View Article][PubMed]
    [Google Scholar]
  7. Cirillo D. M., Heffernan E. J., Wu L., Harwood J., Fierer J., Guiney D. G. 1996; Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion. Infect Immun 64:2019–2023[PubMed]
    [Google Scholar]
  8. Cooke N. M., Smith S. G., Kelleher M., Rogers T. R. 2010; Major differences exist in frequencies of virulence factors and multidrug resistance between community and nosocomial Escherichia coli bloodstream isolates. J Clin Microbiol 48:1099–1104 [View Article][PubMed]
    [Google Scholar]
  9. Czeczulin J. R., Whittam T. S., Henderson I. R., Navarro-Garcia F., Nataro J. P. 1999; Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli . Infect Immun 67:2692–2699[PubMed]
    [Google Scholar]
  10. Dam J., Velikovsky C. A., Mariuzza R. A., Urbanke C., Schuck P. 2005; Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s). Biophys J 89:619–634 [View Article][PubMed]
    [Google Scholar]
  11. Danese P. N., Pratt L. A., Kolter R. 2000; Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol 182:3593–3596[PubMed] [CrossRef]
    [Google Scholar]
  12. Deng Q., Luo W., Donnenberg M. S. 2007; Rapid site-directed domain scanning mutagenesis of enteropathogenic Escherichia coli espD. Biol Proced Online 9:18–26 [View Article][PubMed]
    [Google Scholar]
  13. Evans D. G., Silver R. P., Evans D. J., Chase D. G., Gorbach S. L. 1975; Plasmid-controlled colonization factor associated with virulence in Esherichia coli enterotoxigenic for humans. Infect Immun 12:656–667[PubMed]
    [Google Scholar]
  14. Fagan R. P., Smith S. G. 2007; The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin. FEMS Microbiol Lett 269:248–255 [View Article][PubMed]
    [Google Scholar]
  15. Fagan R. P., Lambert M. A., Smith S. G. 2008; The Hek outer membrane protein of Escherichia coli strain RS218 binds to proteoglycan and utilizes a single extracellular loop for adherence, invasion, and autoaggregation. Infect Immun 76:1135–1142 [View Article][PubMed]
    [Google Scholar]
  16. Fang F. C., Libby S. J., Buchmeier N. A., Loewen P. C., Switala J., Harwood J., Guiney D. G. 1992; The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc Natl Acad Sci U S A 89:11978–11982[PubMed] [CrossRef]
    [Google Scholar]
  17. Fleckenstein J. M., Kopecko D. J., Warren R. L., Elsinghorst E. A. 1996; Molecular characterization of the tia invasion locus from enterotoxigenic Escherichia coli . Infect Immun 64:2256–2265[PubMed]
    [Google Scholar]
  18. Fleckenstein J. M., Holland J. T., Hasty D. L. 2002; Interaction of an uuter membrane protein of enterotoxigenic Escherichia coli with cell surface heparan sulfate proteoglycans. Infect Immun 70:1530–1537 [View Article][PubMed]
    [Google Scholar]
  19. Frick I. M., Mörgelin M., Björck L. 2000; Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein-protein interactions. Mol Microbiol 37:1232–1247[PubMed] [CrossRef]
    [Google Scholar]
  20. Hasman H., Chakraborty T., Klemm P. 1999; Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation. J Bacteriol 181:4834–4841[PubMed]
    [Google Scholar]
  21. Heffernan E. J., Harwood J., Fierer J., Guiney D. 1992a; The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail. J Bacteriol 174:84–91[PubMed] [CrossRef]
    [Google Scholar]
  22. Heffernan E. J., Reed S., Hackett J., Fierer J., Roudier C., Guiney D. 1992b; Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck. J Clin Invest 90:953–964 [View Article][PubMed]
    [Google Scholar]
  23. Heras B., Totsika M., Peters K. M., Paxman J. J., Gee C. L., Jarrott R. J., Perugini M. A., Whitten A. E., Schembri M. A. 2014; The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping. Proc Natl Acad Sci U S A 111:457–462 [View Article][PubMed]
    [Google Scholar]
  24. Herman-Bausier P., El-Kirat-Chatel S., Foster T. J., Geoghegan J. A., Dufrêne Y. F. 2015; Staphylococcus aureus fibronectin-binding protein A mediates cell-cell adhesion through low-affinity homophilic bonds. MBio 6:e0041300415 [View Article][PubMed]
    [Google Scholar]
  25. Klemm P., Hjerrild L., Gjermansen M., Schembri M. A. 2004; Structure-function analysis of the self-recognizing Antigen 43 autotransporter protein from Escherichia coli . Mol Microbiol 51:283–296[PubMed] [CrossRef]
    [Google Scholar]
  26. Klemm P., Vejborg R. M., Sherlock O. 2006; Self-associating autotransporters, SAATs: functional and structural similarities. Int J Med Microbiol 296:187–195 [View Article][PubMed]
    [Google Scholar]
  27. Kolodziejek A. M., Schnider D. R., Rohde H. N., Wojtowicz A. J., Bohach G. A., Minnich S. A., Hovde C. J. 2010; Outer membrane protein X (Ail) contributes to Yersinia pestis virulence in pneumonic plague and its activity is dependent on the lipopolysaccharide core length. Infect Immun 78:5233–5243 [View Article][PubMed]
    [Google Scholar]
  28. Liu Z., Que F., Liao L., Zhou M., You L., Zhao Q., Li Y., Niu H., Wu S. et al. 2014; Study on the promotion of bacterial biofilm formation by a Salmonella conjugative plasmid and the underlying mechanism. PLoS One 9:e109808 [View Article][PubMed]
    [Google Scholar]
  29. Lutwyche P., Rupps R., Cavanagh J., Warren R. A., Brooks D. E. 1994; Cloning, sequencing, and viscometric adhesion analysis of heat-resistant agglutinin 1, an integral membrane hemagglutinin from Escherichia coli O9:H10:K99. Infect Immun 62:5020–5026[PubMed]
    [Google Scholar]
  30. Lutwyche P., Norris-Jones R., Brooks D. E. 1995; Aqueous two-phase polymer systems as tools for the study of a recombinant surface-expressed Escherichia coli hemagglutinin. Appl Environ Microbiol 61:3251–3255[PubMed]
    [Google Scholar]
  31. Mammarappallil J. G., Elsinghorst E. A. 2000; Epithelial cell adherence mediated by the enterotoxigenic Escherichia coli Tia protein. Infect Immun 68:6595–6601[PubMed] [CrossRef]
    [Google Scholar]
  32. Mancini J., Weckselblatt B., Chung Y. K., Durante J. C., Andelman S., Glaubman J., Dorff J. D., Bhargava S., Lijek R. S. et al. 2011; The heat-resistant agglutinin family includes a novel adhesin from enteroaggregative Escherichia coli strain 60A. J Bacteriol 193:4813–4820 [View Article][PubMed]
    [Google Scholar]
  33. Meng G., Spahich N., Kenjale R., Waksman G., St Geme J. W., 3rd. 2011; Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation. EMBO J 30:3864–3874 [View Article][PubMed]
    [Google Scholar]
  34. Menozzi F. D., Boucher P. E., Riveau G., Gantiez C., Locht C. 1994; Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis . Infect Immun 62:4261–4269[PubMed]
    [Google Scholar]
  35. Nataro J. P., Scaletsky I. C., Kaper J. B., Levine M. M., Trabulsi L. R. 1985; Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli . Infect Immun 48:378–383[PubMed]
    [Google Scholar]
  36. Nilson B. H., Frick I. M., Akesson P., Forsén S., Björck L., Akerström B., Wikström M. 1995; Structure and stability of protein H and the M1 protein from Streptococcus pyogenes. Implications for other surface proteins of Gram-positive bacteria. Biochemistry 34:13688–13698 [View Article][PubMed]
    [Google Scholar]
  37. Phillips G. N., Flicker P. F., Cohen C., Manjula B. N., Fischetti V. A. 1981; Streptococcal M protein: alpha-helical coiled-coil structure and arrangement on the cell surface. Proc Natl Acad Sci U S A 78:4689–4693[PubMed] [CrossRef]
    [Google Scholar]
  38. Sambrook J., Russell D. W. 2001 Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  39. Sherlock O., Schembri M. A., Reisner A., Klemm P. 2004; Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation. J Bacteriol 186:8058–8065 [View Article][PubMed]
    [Google Scholar]
  40. Sherlock O., Vejborg R. M., Klemm P. 2005; The TibA adhesin/invasin from enterotoxigenic Escherichia coli is self recognizing and induces bacterial aggregation and biofilm formation. Infect Immun 73:1954–1963 [View Article][PubMed]
    [Google Scholar]
  41. Söding J., Biegert A., Lupas A. N. 2005; The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33:W244–248 [View Article][PubMed]
    [Google Scholar]
  42. Srinivasan U., Foxman B., Marrs C. F. 2003; Identification of a gene encoding heat-resistant agglutinin in Escherichia coli as a putative virulence factor in urinary tract infection. J Clin Microbiol 41:285–289[PubMed] [CrossRef]
    [Google Scholar]
  43. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. 2013; MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729 [View Article][PubMed]
    [Google Scholar]
  44. Torres A. G., Perna N. T., Burland V., Ruknudin A., Blattner F. R., Kaper J. B. 2002; Characterization of Cah, a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli . Mol Microbiol 45:951–966[PubMed] [CrossRef]
    [Google Scholar]
  45. Yachdav G., Kloppmann E., Kajan L., Hecht M., Goldberg T., Hamp T., Hönigschmid P., Schafferhans A., Roos M. et al. 2014; PredictProtein—an open resource for online prediction of protein structural and functional features. Nucleic Acids Res 42:W337–343 [View Article][PubMed]
    [Google Scholar]
  46. Zolkiewski M., Redowicz M. J., Korn E. D., Hammer J. A., Ginsburg A. 1997; Two-state thermal unfolding of a long dimeric coiled-coil: the Acanthamoeba myosin II rod. Biochemistry 36:7876–7883 [View Article][PubMed]
    [Google Scholar]
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