Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Feb;175(3):811–818. doi: 10.1128/jb.175.3.811-818.1993

Neisseria meningitidis produces iron-regulated proteins related to the RTX family of exoproteins.

S A Thompson 1, L L Wang 1, A West 1, P F Sparling 1
PMCID: PMC196221  PMID: 8423153

Abstract

A monoclonal antibody (A4.85) which reacted with Fe-regulated proteins of Neisseria meningitidis, was used to isolate a lambda gt11 clone from N. meningitidis FAM20. Chromosomal fragments flanking the fragment expressing the A4.85 epitope were cloned, and their DNA sequences revealed a 3,345-bp open reading frame predicting a 122-kDa protein. This gene was named frpA (Fe-regulated protein). A computer similarity search of GenBank revealed high levels of similarity to members of the RTX family of cytotoxins, especially in a region of tandem 9-amino-acid repeats. These repeats are found in all members of the RTX family; similar repeats were present 13 times in the predicted FrpA protein. Antigenic relatedness between the meningococcal proteins and the RTX proteins was demonstrated by the reactivity of A4.85 with Escherichia coli hemolysin (HlyA) and Bordetella pertussis adenylate cyclase-hemolysin (CyaA). Similarly, FrpA was recognized by 9D4, a monoclonal antibody directed against B. pertussis CyaA. In addition to the frpA gene, a second gene (frpC) produced a larger RTX-related protein. The frpA and frpC loci were mutagenized in strain FAM20, resulting in the loss of RTX-related proteins. A 120-kDa protein was expressed from the reconstructed frpA gene in E. coli. The biological function of FrpA is unknown, but its similarity to other RTX toxins suggests that it may play an important role in the pathogenesis of meningococcal infection.

Full text

PDF
812

Images in this article

813Image
on p.813
816Image
on p.816
816Image
on p.816

Selected References

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

  1. Biswas G. D., Sox T., Blackman E., Sparling P. F. Factors affecting genetic transformation of Neisseria gonorrhoeae. J Bacteriol. 1977 Feb;129(2):983–992. doi: 10.1128/jb.129.2.983-992.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bjorn M. J., Iglewski B. H., Ives S. K., Sadoff J. C., Vasil M. L. Effect of iron on yields of exotoxin A in cultures of Pseudomonas aeruginosa PA-103. Infect Immun. 1978 Mar;19(3):785–791. doi: 10.1128/iai.19.3.785-791.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black J. R., Dyer D. W., Thompson M. K., Sparling P. F. Human immune response to iron-repressible outer membrane proteins of Neisseria meningitidis. Infect Immun. 1986 Dec;54(3):710–713. doi: 10.1128/iai.54.3.710-713.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bullen J. J., Rogers H. J., Griffiths E. Role of iron in bacterial infection. Curr Top Microbiol Immunol. 1978;80:1–35. doi: 10.1007/978-3-642-66956-9_1. [DOI] [PubMed] [Google Scholar]
  5. Carbonetti N. H., Simnad V. I., Seifert H. S., So M., Sparling P. F. Genetics of protein I of Neisseria gonorrhoeae: construction of hybrid porins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6841–6845. doi: 10.1073/pnas.85.18.6841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang Y. F., Young R., Struck D. K. Cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae. DNA. 1989 Nov;8(9):635–647. doi: 10.1089/dna.1.1989.8.635. [DOI] [PubMed] [Google Scholar]
  7. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  8. Danner D. B. Recovery of DNA fragments from gels by transfer to DEAE-paper in an electrophoresis chamber. Anal Biochem. 1982 Sep 1;125(1):139–142. doi: 10.1016/0003-2697(82)90394-3. [DOI] [PubMed] [Google Scholar]
  9. De Lorenzo V., Herrero M., Giovannini F., Neilands J. B. Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli. Eur J Biochem. 1988 May 2;173(3):537–546. doi: 10.1111/j.1432-1033.1988.tb14032.x. [DOI] [PubMed] [Google Scholar]
  10. DeVoe I. W. The meningococcus and mechanisms of pathogenicity. Microbiol Rev. 1982 Jun;46(2):162–190. doi: 10.1128/mr.46.2.162-190.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dempsey J. A., Litaker W., Madhure A., Snodgrass T. L., Cannon J. G. Physical map of the chromosome of Neisseria gonorrhoeae FA1090 with locations of genetic markers, including opa and pil genes. J Bacteriol. 1991 Sep;173(17):5476–5486. doi: 10.1128/jb.173.17.5476-5486.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dewald B., Baggiolini M. Methods for assessing exocytosis by neutrophil leukocytes. Methods Enzymol. 1986;132:267–277. doi: 10.1016/s0076-6879(86)32014-7. [DOI] [PubMed] [Google Scholar]
  14. Dyer D. W., McKenna W., Woods J. P., Sparling P. F. Isolation by streptonigrin enrichment and characterization of a transferrin-specific iron uptake mutant of Neisseria meningitidis. Microb Pathog. 1987 Nov;3(5):351–363. doi: 10.1016/0882-4010(87)90005-2. [DOI] [PubMed] [Google Scholar]
  15. Dyer D. W., West E. P., McKenna W., Thompson S. A., Sparling P. F. A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-kilodalton iron-regulated protein. Infect Immun. 1988 Apr;56(4):977–983. doi: 10.1128/iai.56.4.977-983.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Felmlee T., Pellett S., Welch R. A. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985 Jul;163(1):94–105. doi: 10.1128/jb.163.1.94-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Felmlee T., Welch R. A. Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5269–5273. doi: 10.1073/pnas.85.14.5269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ferrante A., Thong Y. H. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the Hypaque-Ficoll method. J Immunol Methods. 1980;36(2):109–117. doi: 10.1016/0022-1759(80)90036-8. [DOI] [PubMed] [Google Scholar]
  19. Frey J., Meier R., Gygi D., Nicolet J. Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuropneumoniae. Infect Immun. 1991 Sep;59(9):3026–3032. doi: 10.1128/iai.59.9.3026-3032.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Frey J., Nicolet J. Hemolysin patterns of Actinobacillus pleuropneumoniae. J Clin Microbiol. 1990 Feb;28(2):232–236. doi: 10.1128/jcm.28.2.232-236.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Frey J., Nicolet J. Regulation of hemolysin expression in Actinobacillus pleuropneumoniae serotype 1 by Ca2+. Infect Immun. 1988 Oct;56(10):2570–2575. doi: 10.1128/iai.56.10.2570-2575.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Glaser P., Ladant D., Sezer O., Pichot F., Ullmann A., Danchin A. The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol Microbiol. 1988 Jan;2(1):19–30. [PubMed] [Google Scholar]
  23. Hacker J., Hughes C., Hof H., Goebel W. Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect Immun. 1983 Oct;42(1):57–63. doi: 10.1128/iai.42.1.57-63.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hull S. I., Bieler S., Hull R. A. Restriction fragment length polymorphism and multiple copies of DNA sequences homologous with probes for P-fimbriae and hemolysin genes among uropathogenic Escherichia coli. Can J Microbiol. 1988 Mar;34(3):307–311. doi: 10.1139/m88-056. [DOI] [PubMed] [Google Scholar]
  25. KELLOGG D. S., Jr, PEACOCK W. L., Jr, DEACON W. E., BROWN L., PIRKLE D. I. NEISSERIA GONORRHOEAE. I. VIRULENCE GENETICALLY LINKED TO CLONAL VARIATION. J Bacteriol. 1963 Jun;85:1274–1279. doi: 10.1128/jb.85.6.1274-1279.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kamp E. M., van Leengoed L. A. Serotype-related differences in production and type of heat-labile hemolysin and heat-labile cytotoxin of Actinobacillus (Haemophilus) pleuropneumoniae. J Clin Microbiol. 1989 Jun;27(6):1187–1191. doi: 10.1128/jcm.27.6.1187-1191.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kolodrubetz D., Dailey T., Ebersole J., Kraig E. Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans. Infect Immun. 1989 May;57(5):1465–1469. doi: 10.1128/iai.57.5.1465-1469.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kraft R., Tardiff J., Krauter K. S., Leinwand L. A. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques. 1988 Jun;6(6):544-6, 549. [PubMed] [Google Scholar]
  29. Lally E. T., Golub E. E., Kieba I. R., Taichman N. S., Rosenbloom J., Rosenbloom J. C., Gibson C. W., Demuth D. R. Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins. J Biol Chem. 1989 Sep 15;264(26):15451–15456. [PubMed] [Google Scholar]
  30. Lo R. Y., Strathdee C. A., Shewen P. E. Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica A1. Infect Immun. 1987 Sep;55(9):1987–1996. doi: 10.1128/iai.55.9.1987-1996.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ludwig A., Jarchau T., Benz R., Goebel W. The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes. Mol Gen Genet. 1988 Nov;214(3):553–561. doi: 10.1007/BF00330494. [DOI] [PubMed] [Google Scholar]
  32. Morse S. A., Mietzner T. A., Bolen G., Le Faou A., Schoolnik G. Characterization of the major iron-regulated protein of Neisseria gonorrhoeae and Neisseria meningitidis. Antonie Van Leeuwenhoek. 1987;53(6):465–469. doi: 10.1007/BF00415504. [DOI] [PubMed] [Google Scholar]
  33. Neilands J. B. Microbial envelope proteins related to iron. Annu Rev Microbiol. 1982;36:285–309. doi: 10.1146/annurev.mi.36.100182.001441. [DOI] [PubMed] [Google Scholar]
  34. O'Brien A. D., LaVeck G. D., Thompson M. R., Formal S. B. Production of Shigella dysenteriae type 1-like cytotoxin by Escherichia coli. J Infect Dis. 1982 Dec;146(6):763–769. doi: 10.1093/infdis/146.6.763. [DOI] [PubMed] [Google Scholar]
  35. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pick E. Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. Methods Enzymol. 1986;132:407–421. doi: 10.1016/s0076-6879(86)32026-3. [DOI] [PubMed] [Google Scholar]
  37. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  38. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  39. Salit I. E., Morton G. Adherence of Neisseria meningitidis to human epithelial cells. Infect Immun. 1981 Jan;31(1):430–435. doi: 10.1128/iai.31.1.430-435.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schryvers A. B., Lee B. C. Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae. Can J Microbiol. 1989 Mar;35(3):409–415. doi: 10.1139/m89-063. [DOI] [PubMed] [Google Scholar]
  41. Seifert H. S., Chen E. Y., So M., Heffron F. Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Feb;83(3):735–739. doi: 10.1073/pnas.83.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stephens D. S., McGee Z. A. Attachment of Neisseria meningitidis to human mucosal surfaces: influence of pili and type of receptor cell. J Infect Dis. 1981 Apr;143(4):525–532. doi: 10.1093/infdis/143.4.525. [DOI] [PubMed] [Google Scholar]
  44. Strathdee C. A., Lo R. Y. Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leukotoxin determinant. J Bacteriol. 1989 Feb;171(2):916–928. doi: 10.1128/jb.171.2.916-928.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tai S. P., Holmes R. K. Iron regulation of the cloned diphtheria toxin promoter in Escherichia coli. Infect Immun. 1988 Sep;56(9):2430–2436. doi: 10.1128/iai.56.9.2430-2436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun. 1983 Oct;42(1):33–41. doi: 10.1128/iai.42.1.33-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Welch R. A., Dellinger E. P., Minshew B., Falkow S. Haemolysin contributes to virulence of extra-intestinal E. coli infections. Nature. 1981 Dec 17;294(5842):665–667. doi: 10.1038/294665a0. [DOI] [PubMed] [Google Scholar]
  48. Welch R. A., Falkow S. Characterization of Escherichia coli hemolysins conferring quantitative differences in virulence. Infect Immun. 1984 Jan;43(1):156–160. doi: 10.1128/iai.43.1.156-160.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Welch R. A. Pore-forming cytolysins of gram-negative bacteria. Mol Microbiol. 1991 Mar;5(3):521–528. doi: 10.1111/j.1365-2958.1991.tb00723.x. [DOI] [PubMed] [Google Scholar]
  50. West S. E., Clark V. L. Genetic loci and linkage associations in Neisseria gonorrhoeae and Neisseria meningitidis. Clin Microbiol Rev. 1989 Apr;2 (Suppl):S92–103. doi: 10.1128/cmr.2.suppl.s92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. West S. E., Sparling P. F. Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations. J Bacteriol. 1987 Aug;169(8):3414–3421. doi: 10.1128/jb.169.8.3414-3421.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES