A CYCLOPHILIN-LIKE PEPTIDYL-PROLYL CIS TRANS ISOMERASE FROM LEGIONELLA-PNEUMOPHILA - CHARACTERIZATION, MOLECULAR-CLONING AND OVEREXPRESSION/

Legionella pneumophila is the causative agent of a severe form of pneu monia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBPS5mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we sho w that L. pneumophila Philadelphia I possesses an additional cytoplasm ic PPlase at a level of enzyme activity comparable to that of FKBP25me m. The N-terminal amino acid sequence of the purified protein was obta ined by Edman degradation and showed that the protein is a member of t he cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin ) was cloned and sequenced, It encodes a putative 164-amino-acid prote in with a molecular mass of 17 968 Da called L. pneumophila cyclophili n 18 (L. p. Cyp18), Amino acid sequence comparison displays considerab le similarity to the cytoplasmic and the periplasmic cyclophilins of E scherichia coli with 60.5% and 51.5% identity, respectively. The subst rate specificity and inhibition by cyclosporin A revealed a pattern th at is typically found for other bacterial cyclophilins, An L. pneumoph ila Cyp18 derivative with a 19-amino-acid polypeptide extension includ ing a 6-histidine tag and an enterokinase cleavage site exhibits PPlas e activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant C yp18 were indistinguishable from those of the authentic enzyme. In ord er to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a signi ficant phenotype but was 10-fold less invasive for Acanthamoeba castel lanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activ ity, but this enzymatic activity does not appear to be linked with the native structure of the protein.