Klonierung, Sequenzierung und Charakterisierung der genetischen Determinante des CAMP-Faktors (=cbf-Gen) der Gruppe-B-Streptokokken (Streptococcus agalactiae)

The CAMP reaction, a synergistic lysis of sheep erythrocytes by Staphylococcus aureus (beta-toxin) and group B streptococci (Streptococcus agalactiae) was first described in 1944 by Christie et al.. This phenomenon is caused by the CAMP-factor, an extra-cellular protein which role pathogenicity and virulence is still unknown. The focus of this work is the molecular isolation and characterization of the CAMP-factor gene of group B streptococci (cbf-Gene). First cloning attempts were made using the plasmid pCO63, which resulted from former work of Schneeewind et al. (1984) and produced a protein which reacted with anti-CAMP serum in western blot analysis. After sequencing of the 3,6 kB insert of that clone we found out that it contained a CDP-gycerol hydrolase of E. coli instead of any GBS-DNA. Further on an internal fragment of the cfb gene from group B streptococcal (GBS) strain R268 was amplified by polymerase chain reaction (PCR) using degenerate primers with sequences derived from the CAMP-factor amino acid (aa) sequence of GBS strain NCTC8181 (Ruhlmann et al., 1988). After cloning and sequencing this fragment, the remainder of cfb and the adjacent 5' and 3' sequences were amplified by inverted PCR of genomic DNA and directly sequenced from the PCR product. Within the 1560 bp sequenced, a complete cfb gene deviating in two deduced aa residues from the published sequence was identified. In addition, the cfbR268 sequence contained a 29-aa leader peptide. Using primers directed to the 5' and 3' ends of cfb for PCR, a cfb gene of uniform size could be detected in 19 clinical GBS isolates including three phenotypically CAMP-negative strains. In the CAMP-negative stain 74-360 a possible mutation in the promoter region were found. The complete cfbR268 gene and different portions of its 5' and 3' ends were cloned into the plasmid pJLA602 and expressed in E. coli DH5 alpha. Only the full-sized recombinant CAMP-factor was found to exert co-hemolytic activity in a sheep-blood agar assay.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2005

Author dc:creator

• Blankenstein, Oliver

Contributors dc:contributor

• Lütticken, Rudolf

• info:eu-repo/classification/ddc/610
• Gruppe-B-Streptokokken
• Pathogenitätsfaktor
• CAMP
• CAMP-Faktor
• Protein B
• Streptokokken
• Streptococcus agalactiae
• genetische Determinante
• Medizin

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123723%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:62130