Melanization increased the overall cellular negative charge by 3 to 33% in nine different encapsulated strains of and by 86% in an acapsular strain . led to extensive study of this polymer in spp. . Other species can produce melanin using endogenous compounds or exogenous substrates, and some can produce more than one type of melanin [12,18,19]. Open in a separate window Figure 2 melanin ghosts obtained from lungs Artesunate of infected mice as described in . The red arrows show bud scars on the melanin. In the early 1980s, Kwon-Chung et al. produced melanin-deficient strains (mutants were defective in the active transport system for diphenolic compounds and phenoloxidase, which is the first enzyme needed in Artesunate the L-DOPA melanogenesis pathway. A subsequent study confirmed that the loss of phenoloxidase activity was responsible for the mutant phenotype . Williamson next discovered that laccase, a phenoloxidase, was encoded by the lac1 gene . Laccase was linked to virulence in vivo through disruption of the 5 end of the gene . Interestingly, has two laccase genes, where is 75% similar to lac1, but basal transcript levels of are much lower, and mutation of the gene only induced a mild delay in melanin formation . 3.1. Melanosomes and Melanin Location Melanin synthesis needs to occur in a contained environment as its intermediates are reactive to other surrounding substances . Melanin is synthesized in melanosomes within human melanocytes. In [14,30]. Work in has revealed that disruption of endosomal sorting blocks the deposition of melanin in the cell wall, further supporting non-conventional secretory mechanisms driving proper polymer formation and draws marked similarities to mammalian melanosome biology . The existence of cryptococcal melanosomes explains how melanin is distributed in the cell wall, with the melanosomes depositing diffusely in ring-like layers in the cell wall [19,32]. However, melanins distribution is pathogen-dependent. Whereas has melanin located in the innermost layer of the cell wall , melanin is located on the surface or outer wall layer in spp., spp., spp., [13,34,35,36,37,38]. 3.2. Melanin and Cell Wall Melanins role in the cell wall has been extensively studied in melanin ghosts are made of two to five concentric layers with thicknesses ranging from Artesunate 50 to 75 nm per layer. The layers become thicker as cultures become older . The presence of melanin impacts the cell walls properties. For example, a comparison of WT melanized and non-melanized mutants showed that the conidial wall of the reference strain was composed of several superimposed layers with a thick electron transparent inner layer and two thin electron-dense outer layers, while mutant conidia showed a cell wall devoid of the outermost layer. Scanning electron microscopy (SEM) observed that pigment-less mutants have smooth-walled conidia without ornamentation, and further analysis showed that the mutant cell wall was also less electronegative and hydrophobic . Interestingly, there are still pores present in the cell wall after melanization, which permit the transport of molecules across this barrier. In cell wall pore size also correlated with the age of the culture, as day 4 melanin ghosts displayed the greatest porosity at 41 L ??1 g?1 at 16.4 ?, while day 10 melanin ghosts porosity was 15 L ??1 g?1 at 10 ?. The same FGFR1 study also showed that an antibody against melanin decreased the total porosity from 16 to 8 L ??1 g?1 , which has important implications for drug targeting. In terms of its structure within the cell wall, melanin is described as being composed of irregular granules 50C80 nm in diameter,.