Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1989 Jul;57(7):2050–2056. doi: 10.1128/iai.57.7.2050-2056.1989

Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae.

J Frey 1, J Perrin 1, J Nicolet 1
PMCID: PMC313840  PMID: 2659534

Abstract

The genetic determinant of the cohemolysin which is responsible for the CAMP phenomenon, a cohemolysis, of Actinobacillus pleuropneumoniae was cloned in Escherichia coli. Total DNA from the A. pleuropneumoniae serotype 1 type strain 4074 was used to construct a gene library in plasmid pUC18 in E. coli JM83. A total of 10,500 clones containing recombinant plasmids have been screened for hemolysis on blood plates. Fifty-five clones which showed a weak hemolytic response after 24 to 48 h of incubation were screened for the CAMP reaction with Staphylococcus aureus. This led to the identification of one clone which showed a positive CAMP reaction. Immunoblot analysis revealed that the recombinant strain expressed a protein with a molecular mass of 27,000 daltons, similar in size to the CAMP protein of the group B streptococci. Rabbit antibodies against the CAMP+ clone neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae. Antibodies raised against the cloned CAMP cohemolysin cross-reacted with Streptococcus agalactiae protein B. We designate the 27,000-dalton molecule CAMP factor protein and name its corresponding gene cfp.

Full text

PDF
2050

Images in this article

2052Image
on p.2052
2054Image
on p.2054

Selected References

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

  1. Bendixen P. H., Shewen P. E., Rosendal S., Wilkie B. N. Toxicity of Haemophilus pleuropneumoniae for porcine lung macrophages, peripheral blood monocytes, and testicular cells. Infect Immun. 1981 Sep;33(3):673–676. doi: 10.1128/iai.33.3.673-676.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernheimer A. W., Linder R., Avigad L. S. Nature and mechanism of action of the CAMP protein of group B streptococci. Infect Immun. 1979 Mar;23(3):838–844. doi: 10.1128/iai.23.3.838-844.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. FRASER G. Haemolytic activity of Corynebacterium ovis. Nature. 1961 Jan 21;189:246–246. doi: 10.1038/189246a0. [DOI] [PubMed] [Google Scholar]
  5. Fenwick B. W., Osburn B. I., Olander H. J. Isolation and biological characterization of two lipopolysaccharides and a capsular-enriched polysaccharide preparation from Haemophilus pleuropneumoniae. Am J Vet Res. 1986 Jul;47(7):1433–1441. [PubMed] [Google Scholar]
  6. Figura N., Guglielmetti P. Differentiation of motile and mesophilic Aeromonas strains into species by testing for a CAMP-like factor. J Clin Microbiol. 1987 Jul;25(7):1341–1342. doi: 10.1128/jcm.25.7.1341-1342.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Gunnarsson A., Biberstein E. L., Hurvell B. Serologic studies on porcine strains of Haemophilus parahaemolyticus (pleuropneumoniae): agglutination reactions. Am J Vet Res. 1977 Aug;38(8):1111–1114. [PubMed] [Google Scholar]
  9. Jensen A. E., Bertram T. A. Morphological and biochemical comparison of virulent and avirulent isolates of Haemophilus pleuropneumoniae serotype 5. Infect Immun. 1986 Feb;51(2):419–424. doi: 10.1128/iai.51.2.419-424.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jürgens D., Shalaby F. Y., Fehrenbach F. J. Purification and characterization of cAMP-factor from Streptococcus agalactiae by hydrophobic interaction chromatography and chromatofocusing. J Chromatogr. 1985 Dec 4;348(2):363–370. doi: 10.1016/s0021-9673(01)92474-4. [DOI] [PubMed] [Google Scholar]
  11. Jürgens D., Sterzik B., Fehrenbach F. J. Unspecific binding of group B streptococcal cocytolysin (CAMP factor) to immunoglobulins and its possible role in pathogenicity. J Exp Med. 1987 Mar 1;165(3):720–732. doi: 10.1084/jem.165.3.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kamp E. M., Popma J. K., Van Leengoed L. A. Serotyping of Haemophilus pleuropneumoniae in the Netherlands: with emphasis on heterogeneity within serotype 1 and (proposed) serotype 9. Vet Microbiol. 1987 Mar;13(3):249–257. doi: 10.1016/0378-1135(87)90087-3. [DOI] [PubMed] [Google Scholar]
  13. Kilian M. The haemolytic activity of Haemophilus species. Acta Pathol Microbiol Scand B. 1976 Dec;84B(6):339–341. doi: 10.1111/j.1699-0463.1976.tb01950.x. [DOI] [PubMed] [Google Scholar]
  14. Kume K., Nakai T., Sawata A. Interaction between heat-stable hemolytic substance from Haemophilus pleuropneumoniae and porcine pulmonary macrophages in vitro. Infect Immun. 1986 Feb;51(2):563–570. doi: 10.1128/iai.51.2.563-570.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Köhler W. CAMP-like phenomena of Vibrios. Zentralbl Bakteriol Mikrobiol Hyg A. 1988 Nov;270(1-2):35–40. doi: 10.1016/s0176-6724(88)80139-1. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Linder R., Bernheimer A. W. Enzymatic oxidation of membrane cholesterol in relation to lysis of sheep erythrocytes by corynebacterial enzymes. Arch Biochem Biophys. 1982 Feb;213(2):395–404. doi: 10.1016/0003-9861(82)90565-3. [DOI] [PubMed] [Google Scholar]
  18. MARMUR J., DOTY P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol. 1962 Jul;5:109–118. doi: 10.1016/s0022-2836(62)80066-7. [DOI] [PubMed] [Google Scholar]
  19. Nicolet J. Sur l'hémophilose du pore. 3. Différenciation sérologique de Haemophilus parahaemolyticus. Zentralbl Bakteriol Orig. 1971;216(4):487–495. [PubMed] [Google Scholar]
  20. Nielsen R., O'Connor P. J. Serological characterization of 8 Haemophilus pleuropneumoniae strains and proposal of a new serotype: serotype 8. Acta Vet Scand. 1984;25(1):96–106. doi: 10.1186/BF03547283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nielsen R. Serological characterization of Actinobacillus pleuropneumoniae strains and proposal of a new serotype: serotype 12. Acta Vet Scand. 1986;27(3):453–455. doi: 10.1186/BF03548158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nielsen R. Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 10. Acta Vet Scand. 1985;26(4):581–585. doi: 10.1186/BF03546528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nielsen R. Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9. Acta Vet Scand. 1985;26(4):501–512. doi: 10.1186/BF03546522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nielsen R. Serology of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 strains: establishment of subtypes a and b. Acta Vet Scand. 1986;27(1):49–58. doi: 10.1186/BF03548558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rosendal S., Boyd D. A. Haemophilus pleuropneumoniae serotyping. J Clin Microbiol. 1982 Nov;16(5):840–843. doi: 10.1128/jcm.16.5.840-843.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rühlmann J., Wittmann-Liebold B., Jürgens D., Fehrenbach F. J. Complete amino acid sequence of protein B. FEBS Lett. 1988 Aug 1;235(1-2):262–266. doi: 10.1016/0014-5793(88)81275-4. [DOI] [PubMed] [Google Scholar]
  27. SHOPE R. E. PORCINE CONTAGIOUS PLEUROPNEUMONIA. I. EXPERIMENTAL TRANSMISSION, ETIOLOGY, AND PATHOLOGY. J Exp Med. 1964 Mar 1;119:357–368. doi: 10.1084/jem.119.3.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schneewind O., Friedrich K., Lütticken R. Cloning and expression of the CAMP factor of group B streptococci in Escherichia coli. Infect Immun. 1988 Aug;56(8):2174–2179. doi: 10.1128/iai.56.8.2174-2179.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sebunya T. N., Saunders J. R. Haemophilus pleuropneumoniae infection in swine: a review. J Am Vet Med Assoc. 1983 Jun 15;182(12):1331–1337. [PubMed] [Google Scholar]
  30. Skalka B., Smola J. Lethal effect of CAMP-factor and UBERIS-factor--a new finding about diffusible exosubstances of streptococcus agalactiae and Streptococcus uberis. Zentralbl Bakteriol A. 1981;249(2):190–194. [PubMed] [Google Scholar]
  31. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  34. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES