Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor

Virulence is the outcome of an interaction between the host and a microbe a nd is characterized by a large array of opposing reactions operating at the host-pathogen interface. Cryptococcus neoformans is an important opportuni stic pathogen in immunocompromised patients, including those with human imm unodeficiency virus, and expresses a virulence-associated laccase which is believed to oxidize brain catecholamines and iron as a defense against host immune cells. In the present report, we investigated the cellular location of laccase to understand more fully how it contributes to cryptococcal vir ulence. A monoclonal antibody to the C. neoformans laccase was generated an d used to show localization in the cell walls of representative serotype A (H99) and serotype D (B-3501) strains by immunoelectron microscopy. In addi tion, confocal microscopy was used to show a peripheral location of green f luorescent protein-tagged laccase expressed in live H99 cells. Biochemical studies showed that laccase could be released from intact cells or cell wal l fractions with glucanase enzymes but was retained in the cell wall after sequential extraction with 1 M NaCl, 6 M urea, and 1% sodium dodecyl sulfat e. The presence of a hydrolyzable bond linking laccase to the cell wall was suggested by removal of laccase from cell wall preparations after they wer e boiled in 1% sodium dodecyl sulfate, as was the presence of a disulfide o r thioester bond by removal with dithiothreitol or beta -mercaptoethanol. T hese data show that laccase is present as a tightly associated cell wall en zyme that is readily accessible for interactions with host immune cells.