Cell Wall Targeting of Laccase of Cryptococcus neoformans during Infection of Mice

Abstract

Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1⌬ cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.Cryptococcus neoformans is a basidiomycete fungal pathogen that infects both immunocompetent and immunocompromised individuals (2, 34). This fungus has several characteristics that allow it to survive during pathogenesis, and it produces numerous factors that cause host cell damage. These characteristics and factors include the ability to grow at 37°C, production of a polysaccharide capsule, expression of laccase, urease, and phospholipase, and production of mannitol (4,5,10,27,32,35). The relative importance of each of the characteristics is not fully understood and may be strain dependent; however, the presence of capsule and the presence of laccase are considered major virulence factors.Laccase expression has been correlated with virulence in numerous studies using multiple strains of the fungus (15,29,32). Laccase has been proposed to contribute to virulence through production of melanin pigments and prevention of iron-dependent Fenton products, with resultant accentuation of extrapulmonary dissemination to the brain (14,19,26,36). However, laccase plays little role in pulmonary persistence (26). C. neoformans has two laccase genes in its genome, LAC1 and LAC2, but only LAC1 is expressed significantly under most conditions and deletion of LAC2 results in no reduction in virulence in mice (31, 41). Laccase from C. neoformans is a member of a class of multicopper oxidases that are primarily extracellular proteins expressed in fungi, plants, and insects (24). The main functions in these organisms are polymerization of monolignols to produce the lignin structure of the cell wall of plan...

Cryptococcus neoformans and Cryptococcus gattii, the Etiologic Agents of Cryptococcosis

“…The early observations, using melanin-lacking mutants isolated by UV irradiation, suggesting the importance of melanin as a virulence factor (Kwon- Chung et al 1982c;Rhodes et al 1982) was confirmed by the use of LAC1 gene-deletion mutants (Salas et al 1996). C. neoformans contains two laccase genes, LAC1 and LAC2, in the genome but only LAC1 is expressed significantly under most conditions and virulence is reduced only when the LAC1 gene is deleted (Zhu and Williamson 2004;Pukkila-Worley et al 2005).The cryptococcal laccase, a member of the multicopper oxidases is localized in the cell walls (Zhu et al 2001;Waterman et al 2007b) and its transport to the cell wall is Sec6 dependent (Panepinto et al 2009). Melanization of cryptococci require numerous additional genes such as the copper transporter Ccc2, the copper chaperone Atx1, the chitin synthase Chs3, the transcriptional coactivator Mbf1, the chromatin remodeling enzyme Snf5 (Walton et al 2005), the transcription factor Rim101, and its regulatory gene Rim20 (Liu et al 2008).…”

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

“…Similarly, loss of Vps23 may interrupt the processing and/or transport of laccase (resulting in reduced melanization). Laccase must be loaded with copper during transit through the ER-Golgi compartment network, and it must be properly localized in the cell wall (68). Capsule polysaccharide is also found in extracellular vesicles, although the extent of the contribution of the latter vesicles to capsule formation is not yet clear (67).…”

Copper at the Fungal Pathogen-Host Axis

“…LAC1 mRNA transcription is active in cerebrospinal fluid of C. neoformans-infected rabbits and is an important virulence factor in in vivo models for cryptococcosis (72). Moreover, during brain infection, there is a strong localization of a Lac1-GFP fusion protein at the cryptococcal cell wall, as opposed to the lungs where this enzyme is localized in the cytosol, suggesting a predominant role of the enzyme during the brain infection (73). Several other C. neoformans proteins are also required for melanin biogenesis, such as the Ccc2 Cu ϩ pump that facilitates Cu metallation in the secretory compartment and is known to be a virulence factor in murine infection models (33).…”