A cyclophilin‐like peptidyl‐prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Abstract

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order 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 significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

Recombinant Cyclophilins Lack Nuclease Activity

“…1, center panel, lane H). A previous report showed that, at least for the L. pneumophila LpCyp18 cyclophilin, the hypothetical associated nuclease activity was not linked with its native structure (12). These results are coherent with our find-ing that the nuclease activity is caused by a different protein.…”

“…Some reports on human recombinant CypA, CypB, and CypC cyclophilins (6), murine CypB cyclophilin (8), Legionella pneumophila LpCyp18 cyclophilin (12), and Streptomyces antibioticus-SanCyp18 cyclophilin (9) describe associated Ca 2ϩ / Mg 2ϩ nuclease-dependent activity. For the aforementioned eukaryotic cyclophilins, the accompanying nuclease activity has been proposed as playing a central role in apoptosis.…”

“…We used several E. coli strains as hosts to test the putative nuclease activity for recombinant human CypA (7) and recombinant LpCyp18 (12) cyclophilin. E. coli NM522 and M15 strains were used in the studies mentioned above to express human CypA and LpCyp18, respectively (7,12). Both of them possess the aforementioned periplasmic endonuclease I, which was absent from the E. coli JM109 strain used to express SanCyp18.…”

“…1, center panel, lane C). We expressed human CypA and LpCyp18 cyclophilins genes in E. coli M15 (pREP4) (endA ϩ ) and E. coli JM105 (pREP4) (endA mutant) (14) by the use of pTTE1 expression vector (12) for the LpCyp18 gene and pTTE2 expression vector (Thomas Tradler, Max-Planck Forschungsstelle "Enzymologie der Proteinfaltung," Halle/Saale, Germany) for the human CypA gene to investigate the hypothetical associated nuclease activity. The plasmids were introduced in the aforementioned strains to overexpress these proteins, following the protocols described for the QIAGEN system employed.…”

Histoplasma capsulatumCyclophilin A Mediates Attachment to Dendritic Cell VLA-5

“…Legionella also possess a second cytoplasmic cyclophilin, cyclophilin 18. Mip protein is not necessary for the extracellular survival of Legionella, but disruption mutants are 10 times less invasive of Acanthamoeba castellani (20). Interestingly, the Hc CypA sequence is not homologous to L. pneumophila Mip, but shows a high degree of conservation with the cytoplasmic form cyclophilin 18.…”