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

Publication ,  Journal Article
Salas, SD; Bennett, JE; Kwon-Chung, KJ; Perfect, JR; Williamson, PR
Published in: J Exp Med
August 1, 1996

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-directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.

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Published In

J Exp Med

DOI

ISSN

0022-1007

Publication Date

August 1, 1996

Volume

184

Issue

2

Start / End Page

377 / 386

Location

United States

Related Subject Headings

  • Rabbits
  • RNA, Messenger
  • RNA, Fungal
  • Oxidoreductases
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Mice, Inbred BALB C
  • Mice
  • Melanins
  • Laccase
 

Citation

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Salas, S. D., Bennett, J. E., Kwon-Chung, K. J., Perfect, J. R., & Williamson, P. R. (1996). Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med, 184(2), 377–386. https://doi.org/10.1084/jem.184.2.377
Salas, S. D., J. E. Bennett, K. J. Kwon-Chung, J. R. Perfect, and P. R. Williamson. “Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.J Exp Med 184, no. 2 (August 1, 1996): 377–86. https://doi.org/10.1084/jem.184.2.377.
Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med. 1996 Aug 1;184(2):377–86.
Salas, S. D., et al. “Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.J Exp Med, vol. 184, no. 2, Aug. 1996, pp. 377–86. Pubmed, doi:10.1084/jem.184.2.377.
Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med. 1996 Aug 1;184(2):377–386.

Published In

J Exp Med

DOI

ISSN

0022-1007

Publication Date

August 1, 1996

Volume

184

Issue

2

Start / End Page

377 / 386

Location

United States

Related Subject Headings

  • Rabbits
  • RNA, Messenger
  • RNA, Fungal
  • Oxidoreductases
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Mice, Inbred BALB C
  • Mice
  • Melanins
  • Laccase