title: Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans.
abstract: Previous studies have shown that a Deltavph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper-containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Deltavph1 mutant as shown by Western blot and immuno-electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Deltavph1 cells at 30 degrees C by addition of 100 micro M copper sulphate. This restoration by copper was found to occur through both transcriptional and post-translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5'-untranslated region of CNLAC1. Copper was also found to restore partial activity to Deltavph1 cells at 0 degrees C, suggesting that cell wall laccase was expressed in the mutant as an apo-enzyme. Apo-laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)-ATPase proton pump in copper assembly of laccase in C. neoformans.
authors.affiliation: Division of Infectious Diseases, and University of Illinois at Chicago College of Medicine, Chicago, IL 60612, USA.
authors.forename: Xudong
authors.initials: X
authors.lastname: Zhu
authors.forename: Jack
authors.initials: J
authors.lastname: Gibbons
authors.forename: Shirong
authors.initials: S
authors.lastname: Zhang
authors.forename: Peter R
authors.initials: PR
authors.lastname: Williamson
mesh_terms: D000818:Animals; D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology / Q000648:ultrastructure; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D004271:DNA, Fungal / Q000235:genetics; D042845:Laccase; D051379:Mice; D016253:Microscopy, Immunoelectron; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D014158:Transcription, Genetic / Q000187:drug effects; D014774:Virulence / Q000235:genetics
chemical_list: D001051:Apoenzymes; D004271:DNA, Fungal; D003300:Copper; D010088:Oxidoreductases; D042845:Laccase