Laccase of Cryptococcus neoformans Is a Cell Wall-Associated Virulence Factor

Abstract

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 ␤-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.

Vesicle-associated melanization in Cryptococcus neoformans

“…Previous studies suggested that melanization occurred when laccase in the cell wall oxidized a substrate, such as L-DOPA, ultimately resulting in polymerization of melanin (Waterman et al, 2007;Zhu et al, 2001). However, recent studies showing the presence of cell-wall-associated vesicles exhibiting laccase activity raised the possibility of the involvement of vesicles in melanization (Rodrigues et al, 2008).…”

“…Melanization of C. neoformans depends on the presence of substrate, such as L-3,4-dihydroxyphenylalanine (L-DOPA) and other catecholamines (Garcia-Rivera et al, 2005). In addition, it requires expression of laccase, a cell wall enzyme that is associated with virulence (Zhu et al, 2001). In melanized cells, melanin is found in the cell wall of the yeast .…”

Laccase: enzyme revisited and function redefined

“…B. subtilis endospore coat protein shows CotA oxidase activity with all the structural features of laccase, including the reactive surface exposed copper center T1 and two buried copper centers (T2 and T3) [29]. Cot A is naturally associated with the coat structure in active form, which suggests B. subtilis endospore coat structure as a surface display system for biocatalyst applications involving the stable CotA laccase [38]. Comparison of B. subtilis CotA with other multicopper oxidases shows that copper binding motives are conserved in all sequences.…”

“…Recombinant cryptococcal laccase exhibits iron oxidase activity by converting Fe (II) to Fe (III) [27]. Moreover, laccase from C. neoformans is implicated in the virulence of the organism by virtue of its oxidizing action on the brain catecholamines as a defense system against host immune system [2,29]. Its differential function when secreted by different type of cells adds it to the moonlighting list (Fig.…”

“…These proteins are assembled into a lamella-like inner coat and an electron-dense outer coat, which protects the spore from a diverse range of stress [37]. B. subtilis endospore coat protein shows CotA oxidase activity with all the structural features of laccase, including the reactive surface exposed copper center T1 and two buried copper centers (T2 and T3) [29]. Cot A is naturally associated with the coat structure in active form, which suggests B. subtilis endospore coat structure as a surface display system for biocatalyst applications involving the stable CotA laccase [38].…”