Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Oct;65(10):4199–4206. doi: 10.1128/iai.65.10.4199-4206.1997

Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes.

Y T Pan 1, B Xu 1, K Rice 1, S Smith 1, R Jackson 1, A D Elbein 1
PMCID: PMC175603  PMID: 9317027

Abstract

Enterobacter cloacae has been implicated as one of the causative agents in neonatal infection and causes a septicemia thought to be initiated via the gastrointestinal tract. The adhesion of radiolabeled E. cloacae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrations of alpha-mannosides and micromolar concentrations of high-mannose oligosaccharides. A variety of well-characterized mannose oligosaccharides were tested as inhibitors of adhesion. The best inhibitor was the Man9(GlcNAc)2-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man7(GlcNAc)2, Man6(GlcNAc)2 or Man5(GlcNAc)2. Further evidence that the bacteria preferred Man9(GlcNAc)2 structures was obtained by growing HT-29 cells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man9(GlcNAc)2 at the cell surface. Such cells bound 1.5- to 2-fold more bacteria than did control cells. The adhesin involved in binding to high-mannose structures was purified from isolated pili. On sodium dodecyl sulfate-gels, a 35-kDa protein was identified by its specific binding to a mannose-containing biotinylated albumin. The amino acid sequences of several peptides from the 35-kDa subunit showed over 85% identity to FimH, the mannose-specific adhesin of Salmonella typhimurium. Pili were labeled with 125I and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man9(GlcNAc)2-tyrosinamide but not by oligosaccharides with fewer mannose residues. Polyclonal antibody against this 35-kDa protein also effectively blocked adhesion of pili or E. cloacae, but no effect was observed with nonspecific antibody. These studies demonstrate that the 35-kDa pilus subunit is a lectin whose specificity is directed toward Man, (GlcNAc)2 oligosaccharides.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Acolet D., Ahmet Z., Houang E., Hurley R., Kaufmann M. E. Enterobacter cloacae in a neonatal intensive care unit: account of an outbreak and its relationship to use of third generation cephalosporins. J Hosp Infect. 1994 Dec;28(4):273–286. doi: 10.1016/0195-6701(94)90091-4. [DOI] [PubMed] [Google Scholar]
  2. Beachey E. H. Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surface. J Infect Dis. 1981 Mar;143(3):325–345. doi: 10.1093/infdis/143.3.325. [DOI] [PubMed] [Google Scholar]
  3. Clegg S., Pruckler J., Purcell B. K. Complementation analyses of recombinant plasmids encoding type 1 fimbriae of members of the family Enterobacteriaceae. Infect Immun. 1985 Oct;50(1):338–340. doi: 10.1128/iai.50.1.338-340.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Corradi Da Silva M. L., Stubbs H. J., Tamura T., Rice K. G. 1H NMR characterization of a hen ovalbumin tyrosinamide N-linked oligosaccharide library. Arch Biochem Biophys. 1995 Apr 20;318(2):465–475. doi: 10.1006/abbi.1995.1255. [DOI] [PubMed] [Google Scholar]
  5. Elbein A. D. Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing. FASEB J. 1991 Dec;5(15):3055–3063. doi: 10.1096/fasebj.5.15.1743438. [DOI] [PubMed] [Google Scholar]
  6. Elbein A. D., Solf R., Dorling P. R., Vosbeck K. Swainsonine: an inhibitor of glycoprotein processing. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7393–7397. doi: 10.1073/pnas.78.12.7393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elbein A. D., Tropea J. E., Mitchell M., Kaushal G. P. Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I. J Biol Chem. 1990 Sep 15;265(26):15599–15605. [PubMed] [Google Scholar]
  8. Fader R. C., Duffy L. K., Davis C. P., Kurosky A. Purification and chemical characterization of type 1 pili isolated from Klebsiella pneumoniae. J Biol Chem. 1982 Mar 25;257(6):3301–3305. [PubMed] [Google Scholar]
  9. Firon N., Ashkenazi S., Mirelman D., Ofek I., Sharon N. Aromatic alpha-glycosides of mannose are powerful inhibitors of the adherence of type 1 fimbriated Escherichia coli to yeast and intestinal epithelial cells. Infect Immun. 1987 Feb;55(2):472–476. doi: 10.1128/iai.55.2.472-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Firon N., Ofek I., Sharon N. Carbohydrate-binding sites of the mannose-specific fimbrial lectins of enterobacteria. Infect Immun. 1984 Mar;43(3):1088–1090. doi: 10.1128/iai.43.3.1088-1090.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gallagher P. G. Enterobacter bacteremia in pediatric patients. Rev Infect Dis. 1990 Sep-Oct;12(5):808–812. doi: 10.1093/clinids/12.5.808. [DOI] [PubMed] [Google Scholar]
  12. Giampapa C. S., Abraham S. N., Chiang T. M., Beachey E. H. Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes. J Biol Chem. 1988 Apr 15;263(11):5362–5367. [PubMed] [Google Scholar]
  13. Gladstone I. M., Ehrenkranz R. A., Edberg S. C., Baltimore R. S. A ten-year review of neonatal sepsis and comparison with the previous fifty-year experience. Pediatr Infect Dis J. 1990 Nov;9(11):819–825. doi: 10.1097/00006454-199011000-00009. [DOI] [PubMed] [Google Scholar]
  14. Hanson M. S., Brinton C. C., Jr Identification and characterization of E. coli type-1 pilus tip adhesion protein. Nature. 1988 Mar 17;332(6161):265–268. doi: 10.1038/332265a0. [DOI] [PubMed] [Google Scholar]
  15. Hanson M. S., Hempel J., Brinton C. C., Jr Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein. J Bacteriol. 1988 Aug;170(8):3350–3358. doi: 10.1128/jb.170.8.3350-3358.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heusser M. F., Patterson J. E., Kuritza A. P., Edberg S. C., Baltimore R. S. Emergence of resistance to multiple beta-lactams in Enterobacter cloacae during treatment for neonatal meningitis with cefotaxime. Pediatr Infect Dis J. 1990 Jul;9(7):509–512. doi: 10.1097/00006454-199007000-00011. [DOI] [PubMed] [Google Scholar]
  17. Hornick D. B., Allen B. L., Horn M. A., Clegg S. Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae. J Clin Microbiol. 1991 Sep;29(9):1795–1800. doi: 10.1128/jcm.29.9.1795-1800.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones C. H., Pinkner J. S., Roth R., Heuser J., Nicholes A. V., Abraham S. N., Hultgren S. J. FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2081–2085. doi: 10.1073/pnas.92.6.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Karpuch J., Goldberg M., Kohelet D. Neonatal bacteremia. A 4-year prospective study. Isr J Med Sci. 1983 Nov;19(11):963–966. [PubMed] [Google Scholar]
  20. Keith B. R., Maurer L., Spears P. A., Orndorff P. E. Receptor-binding function of type 1 pili effects bladder colonization by a clinical isolate of Escherichia coli. Infect Immun. 1986 Sep;53(3):693–696. doi: 10.1128/iai.53.3.693-696.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kohno K., Yamamoto T., Kuroiwa A., Amako K. Purification and characterization of Serratia marcescens US5 pili. Infect Immun. 1984 Nov;46(2):295–300. doi: 10.1128/iai.46.2.295-300.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Korhonen T. K., Lounatmaa K., Ranta H., Kuusi N. Characterization of type 1 pili of Salmonella typhimurium LT2. J Bacteriol. 1980 Nov;144(2):800–805. doi: 10.1128/jb.144.2.800-805.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  24. Krogfelt K. A. Bacterial adhesion: genetics, biogenesis, and role in pathogenesis of fimbrial adhesins of Escherichia coli. Rev Infect Dis. 1991 Jul-Aug;13(4):721–735. doi: 10.1093/clinids/13.4.721. [DOI] [PubMed] [Google Scholar]
  25. Krogfelt K. A., Bergmans H., Klemm P. Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae. Infect Immun. 1990 Jun;58(6):1995–1998. doi: 10.1128/iai.58.6.1995-1998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lis H., Sharon N., Katchalski E. Soybean hemagglutinin, a plant glycoprotein. I. Isolation of a glycopeptide. J Biol Chem. 1966 Feb 10;241(3):684–689. [PubMed] [Google Scholar]
  27. Lis H., Sharon N. Soybean agglutinin--a plant glycoprotein. Structure of the carboxydrate unit. J Biol Chem. 1978 May 25;253(10):3468–3476. [PubMed] [Google Scholar]
  28. Markwell M. A. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Anal Biochem. 1982 Sep 15;125(2):427–432. doi: 10.1016/0003-2697(82)90025-2. [DOI] [PubMed] [Google Scholar]
  29. Minion F. C., Abraham S. N., Beachey E. H., Goguen J. D. The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit. J Bacteriol. 1986 Mar;165(3):1033–1036. doi: 10.1128/jb.165.3.1033-1036.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Molyneux R. J., James L. F. Loco intoxication: indolizidine alkaloids of spotted locoweed (Astragalus lentiginosus). Science. 1982 Apr 9;216(4542):190–191. doi: 10.1126/science.6801763. [DOI] [PubMed] [Google Scholar]
  31. Neeser J. R., Koellreutter B., Wuersch P. Oligomannoside-type glycopeptides inhibiting adhesion of Escherichia coli strains mediated by type 1 pili: preparation of potent inhibitors from plant glycoproteins. Infect Immun. 1986 May;52(2):428–436. doi: 10.1128/iai.52.2.428-436.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ofek I., Mosek A., Sharon N. Mannose-specific adherence of Escherichia coli freshly excreted in the urine of patients with urinary tract infections, and of isolates subcultured from the infected urine. Infect Immun. 1981 Dec;34(3):708–711. doi: 10.1128/iai.34.3.708-711.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ofek I., Sharon N. Adhesins as lectins: specificity and role in infection. Curr Top Microbiol Immunol. 1990;151:91–113. doi: 10.1007/978-3-642-74703-8_5. [DOI] [PubMed] [Google Scholar]
  34. Orndorff P. E., Falkow S. Organization and expression of genes responsible for type 1 piliation in Escherichia coli. J Bacteriol. 1984 Aug;159(2):736–744. doi: 10.1128/jb.159.2.736-744.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pan Y. T., Hori H., Saul R., Sanford B. A., Molyneux R. J., Elbein A. D. Castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin. Biochemistry. 1983 Aug 2;22(16):3975–3984. doi: 10.1021/bi00285a038. [DOI] [PubMed] [Google Scholar]
  36. Rodriguez-Ortega M., Ofek I., Sharon N. Membrane glycoproteins of human polymorphonuclear leukocytes that act as receptors for mannose-specific Escherichia coli. Infect Immun. 1987 Apr;55(4):968–973. doi: 10.1128/iai.55.4.968-973.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. SEIFTER S., DAYTON S. The estimation of glycogen with the anthrone reagent. Arch Biochem. 1950 Jan;25(1):191–200. [PubMed] [Google Scholar]
  38. Salit I. E., Gotschlich E. C. Hemagglutination by purified type I Escherichia coli pili. J Exp Med. 1977 Nov 1;146(5):1169–1181. doi: 10.1084/jem.146.5.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Svambprg Edén C., Andersson B., Aniansson G., Lindstedt R., de Man P., Nielsen A., Leffler H., Wold A. Inhibition of bacterial attachment: examples from the urinary and respiratory tracts. Curr Top Microbiol Immunol. 1990;151:167–184. doi: 10.1007/978-3-642-74703-8_9. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES