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

Figures & data

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).

Table 1 Primers used for real-time PCR amplification

Locus	5′ primer	3′ Primer
CNB04110	GTC GTC ATT TCG GCC ATT	GAC CAG GGA TGC TGA TTT CT
CNK02300	AAC ACT GGA TTG ATC CGA CA	CTT GAT GTG GTC GAA ATT GG
CND02380	GCT GAC CGA GTC GGC GTC	GAC GAG AGT CAC CGG TAG TG
CND03820	TGC GTT TGT TAC TGA AAC CC	GCA TCC TCC TCT TCC TCT TG
CNG04630	GAG CGC TAC ATT CC T GAT GA	GAA GCG AAT GCT AGT GAG TAG G
CNB04240	GTT GGC AAG TTT GTT TCC CT	ACC AAC AGA GGG CTC AGA GT

Table 2 Fold changes in gene expression upon addition of L-DOPA to cultures

Locus	Putative function	Microarray		Real-Time PCR
		Wild-type	Wild-type	LAC1 deletion	Complemented
CNB04110	Isoflavone reductase	12.9	16.6	0.85	4.2
CNK02300	Glutathione transferase	3.9	4.2	2.2	40
CND02380	NADH flavin oxidoreductase	2.7	1.6	1.5	4
CND03820	Myosin I binding protein	2.1	2.5	0.77	1
CNG04630	Permease	2.1	4.8	0.86	1.4
CNB04240	Benzoquinone oxidoreductase	2.0	2.1	0.87	1.6
CND00360	Unknown	6.7		Undetectable
CNM01700	Unknown	2.0		Not determined