Function and secretion of Cryptococcus neoformans virulence factors glucuronoxylomannan and laccase

Abstract

Cryptococcus neoformans is an environmental yeast and the etiologic agent of cryptococcosis. This thesis is focused on the secretion and function of two C. neoformans virulence factors: capsule and laccase. CAP59 is a capsule-related gene of unknown function. cap59Delta (C536) is an acapsular strain as observed by India ink and immunofluorescence. Despite being acapsular, C536 makes capsule material as shown by capture ELISA and confirmed by immunogold labeling with monoclonal antibodies (mAbs). C536 failed to degrade raffinose and urea and cells had abnormal size. Laccase is a phenoloxidase that allows C. neoformans to polymerize phenolic compounds to melanin and contributes to virulence. Two mAbs to laccase were generated, G3P4D3 (gamma1kappa) and G3P2H11 (gamma1kappa). Expression of laccase in vivo was shown by immunohistochemistry and immunogold labeling. Quantification of laccase labeling showed a shift in localization from the cytoplasm/cell wall to the capsule during the course of infection. Laccase was also found in culture supernatant in vitro . The molecular weight of secreted laccase was larger in cap59Delta, a difference that was attributed to glycosylation. Our results suggest that CAP59 is involved in regulation of glycosylation which can affect cellular trafficking. We also evaluated the susceptibility of melanized C. neoformans to silver nitrate. Melanized cells were less susceptible to toxic concentrations of silver nitrate and pre-incubation of silver nitrate solutions with melanin ghost reduced the toxicity of the solution to unmelanized cells. Protection by melanin particles was shown to be mediated by the binding of silver ion to melanin reducing susceptibility of C. neoformans. C. neoformans can only make melanin using exogenous substrates, such as L-dopa, MethylDOPA, Epinephrine and Norepinephrine. Cells grown in Norepinephrine were smaller and had lower zeta potential than the cells grown in other substrates. Electron spin resonance analysis of Epinephrine did not show the classical signal associated with stable free radical population. In addition, not all the melanin ghost derived from Epinephrine reacted with mAb 6D2. These results associate the function of cap59 gene with the proper expression of two virulence genes and provide new insights into the function of melanin.