TY - JOUR
T1 - Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor
AU - Zhu, X.
AU - Gibbons, J.
AU - Garcia-Rivera, J.
AU - Casadevall, A.
AU - Williamson, P. R.
PY - 2001
Y1 - 2001
N2 - Virulence is the outcome of an interaction between the host and a microbe and is characterized by a large array of opposing reactions operating at the host-pathogen interface. Cryptococcus neoformans is an important opportunistic pathogen in immunocompromised patients, including those with human immunodeficiency 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 virulence. A monoclonal antibody to the C. neoformans laccase was generated and used to show localization in the cell walls of representative serotype A (H99) and serotype D (B-3501) strains by immunoelectron microscopy. In addition, confocal microscopy was used to show a peripheral location of green fluorescent protein-tagged laccase expressed in live H99 cells. Biochemical studies showed that laccase could be released from intact cells or cell wall 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 sulfate. The presence of a hydrolyzable bond linking laccase to the cell wall was suggested by removal of laccase from cell wall preparations after they were boiled in 1% sodium dodecyl sulfate, as was the presence of a disulfide or thioester bond by removal with dithiothreitol or β-mercaptoethanol. These data show that laccase is present as a tightly associated cell wall enzyme that is readily accessible for interactions with host immune cells.
AB - Virulence is the outcome of an interaction between the host and a microbe and is characterized by a large array of opposing reactions operating at the host-pathogen interface. Cryptococcus neoformans is an important opportunistic pathogen in immunocompromised patients, including those with human immunodeficiency 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 virulence. A monoclonal antibody to the C. neoformans laccase was generated and used to show localization in the cell walls of representative serotype A (H99) and serotype D (B-3501) strains by immunoelectron microscopy. In addition, confocal microscopy was used to show a peripheral location of green fluorescent protein-tagged laccase expressed in live H99 cells. Biochemical studies showed that laccase could be released from intact cells or cell wall 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 sulfate. The presence of a hydrolyzable bond linking laccase to the cell wall was suggested by removal of laccase from cell wall preparations after they were boiled in 1% sodium dodecyl sulfate, as was the presence of a disulfide or thioester bond by removal with dithiothreitol or β-mercaptoethanol. These data show that laccase is present as a tightly associated cell wall enzyme that is readily accessible for interactions with host immune cells.
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U2 - 10.1128/IAI.69.9.5589-5596.2001
DO - 10.1128/IAI.69.9.5589-5596.2001
M3 - Article
C2 - 11500433
AN - SCOPUS:0034867481
SN - 0019-9567
VL - 69
SP - 5589
EP - 5596
JO - Infection and immunity
JF - Infection and immunity
IS - 9
ER -