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

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.

The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins

“…The finding that CAMP activity of serotype 1 is solely caused by ApxI and ApxII demonstrates unequivocally that the cloned cfp gene cannot be responsible for the CAMP effect of A. pleuropneumoniae serotype 1 [7]. This is in support of the suggestions of MacInnes et al and Green et al [8,9] that the cfi gene codes for a regulatory protein that activates a latent haemolysin in E. coli.…”

“…The data of Devenish et al with respect to the serotypes 2 and 6 do not support our hypothesis. However, the cytotoxic and haemolytic activities of the serotypes 2 and 6 strains that were used differed from the activities reported by others for these serotypes [3,7,11].…”

“…This synergistic haemolysis of sheep erythrocytes is caused by a haemolytic compound of A. pleuropneumoniae and a phospholipase or sphingomyelinase produced by S. aureus. The latter enzymes hydrolyze phospholipids from the erythrocyte membrane and make these cells more susceptible to haemolysis [7]. The CAMP effect is displayed by several bacterial species and is an important tool for taxonomic classifications.…”

“…Besides its use in diagnosis, the putative CAMP factor of A. pleuropneumoniae drew attention as a virulence factor. Frey et al identified a gene from A. pleuropneumoniae that conferred CAMP activity on E. coli [7]. This C$LJ gene coded for a protein of 27 kDa, which was supposed to be the CAMP factor protein (CFP) of A. pleuropneumoniae [7].…”

“…Frey et al identified a gene from A. pleuropneumoniae that conferred CAMP activity on E. coli [7]. This C$LJ gene coded for a protein of 27 kDa, which was supposed to be the CAMP factor protein (CFP) of A. pleuropneumoniae [7]. MacInnes et al described the hlyX gene of A. pleuropneumoniae that coded for the HlyX protein of 27.1 kDa.…”