The effect of L-DOPA on Cryptococcus neoformans growth and gene expression

Abstract

Cryptococcus neoformans is unusual among melanotic fungi in that it requires an exogenous supply of precursor to synthesize melanin. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. L-3,4-dihydroxyphenylalanine (L-DOPA) is a natural catecholamine that is frequently used to induce melanization in C. neoformans and L-DOPA-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents and heavy metals. Given that C. neoformans needs exogenous substrate for melanization one question in the field is the extent to which melanin-associated phenotypes reflect the presence of melanin or metabolic changes in response to substrates. In this study we analyze the response of C. neoformans to L-DOPA with respect to melanization, gene expression and metabolic incorporation. Increasing the concentration of L-DOPA promotes melanin formation up to concentrations > 1 mM, after which toxicity is apparent as manifested by reduced growth. The timing of C. neoformans cells to melanization is affected by growth phase and cell density. Remarkably, growth of C. neoformans in the presence of L-DOPA results in the induction of relatively few genes, most of which could be related to stress metabolism. We interpret these results to suggest that the biological effects associated with melanization after growth in L-DOPA are largely due to the presence of the pigment. This in turn provides strong support for the view that melanin contributes to virulence directly through its presence in the cell wall.

Figure 1

Melanization dose response of C. neoformans to L-DOPA and Norepinephrine. Cells of strains JEC 21 (top row) and H99 (bottom row) were serially diluted and plated on media with or without the indicated concentration of substrate (mM). Plates were incubated for four (L-DOPA) or seven (Norepinephrine) days before photographing.

Figure 2

Effect of culture age on the rate of L-DOPA-mediated melanization. “New” indicates L-DOPA was added to cultures at the time of inoculation and incubated two days. “Old” indicates L-DOPA was added to a seven-day culture of C. neoformans and incubated for one or two more days. Left tube, strain JEC 21; right tube, strain H99.

Figure 3

Effect of cell density on melanization. (A) C. neoformans strain JEC 21 was grown overnight in chemically defined minimal medium and concentrated before adding L-DOPA. Flasks were photographed after overnight incubation with L-DOPA. Cell densities of the cultures before adding L-DOPA were 6.6 × 10⁵, 4.6 × 10⁶ and 3.6 × 10⁷ CFU/ml. (B) L-DOPA agar was inoculated with 12, 135 or 1,250 CFUs of C. neoformans strain JEC 21 (top) or 14, 90 and 1,400 CFU of strain H99 (bottom) and incubated seven days at 30°C.

Figure 4

Fold changes in gene expression over time. C. neoformans wild-type (top) and laccase deletion (bottom) cultures were incubated with or without L-DOPA for 1–8 days. RNA was isolated from the cells at the indicated times for gene expression analysis by real-time PC R. The experiment was repeated twice. The results of one representative time course are shown. The fold changes in gene expression in the presence of L-DOPA are indicated for each gene.

Figure 5

Incorporation of L-DOPA by cells. Assays were performed as described in the Methods and the percentage of L-DOPA incorporated calculated at indicated time intervals. Strains used were JEC 21 (WT), 2E-TU (Δlac1), 2E-TUC (COMPL.) and heated killed JEC 21 (HK).