Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.

作者：Stephanie Salas, John E. Bennett, K. J. Kwon-Chung, John R. Perfect, Peter Richard Williamson · 发表于：The Journal of Experimental Medicine · 年份：1996 · DOI：10.1084/jem.184.2.377 · 被引用次数：411 · 研究领域：Fungal Infections and Studies、Antifungal resistance and susceptibility、Nail Diseases and Treatments

To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper-binding site. When this base change was introduced into CNLAC1 by site-di...