Table 1.
Optimum conditions for the production of the extracellular pink and intracellular brown pigments formed by Cryptococcus spp. from L-tryptophan.
Figure 1.
Pink and brown pigment production by Cryptococcus.
The intensity of the pink and brown pigments were estimated visually. Strong pink pigment was produced in m-LTG media by C.neoformans var. grubii (A) C. gattii (B) formed light brown pigments on quadrants I and IV and C. neoformans var. neoformans and C. neoformans var. grubii produced pink pigments (quadrants II and III) respectively on m-LTG agar. The intensity of the pink pigment varied from strain to strain. Some strains of C. neoformans var. neoformans produced brown pigments on m-LTG agar (not shown). C. gattii produced an intense brown pigment after growth in m-FLTG medium (C).
Table 2.
Comparison of pigment production from L- and D-Tryptophan by Cryptococcus spp.
Figure 2.
Pink pigment spectra from 200 to 760 nm.
The scan at pH 5 of the purified pink pigment revealed two peaks in the UV range (231 and 265 nm). A single peak was seen in the visible range (538 nm). The scan at pH 1 shows a shift in the UV peaks to (250 and possible 260 nm) and a single enhanced peak (at 538 nm). The scan at pH 12 reveals a UV peak at 243 nm which had a large shoulder and the peak at 538 nm has disappeared. The disappearance of the peak at 538 nm corresponded to the pink pigment becoming colorless above the pH of 6.0.
Figure 3.
Visible and fluorescent microscopy of Cryptococcus.
Visible and fluorescent microscopy (FITC filter) of C. gattii, NIH 112, (top) and C. neoformans var. grubii, H99, (bottom). The majority of the yeast cells have a normal appearance but atypical morphology was occasionally observed after growth on tryptophan. C. gattii and C. neoformans var. grubii cells exhibit strong fluorescence with the FITC filter. C. gattii cells often contained small concentrated foci that were fluorescent. The cell walls of C. neoformans var. grubii were usually fluorescent. The strains of C. neoformans var. neoformans which produced brown pigments exhibited a fluorescent pattern that resembled C. gattii whereas the strains that produced the pink pigment had a fluorescent pattern that was similar to C. neoformans var. grubii.
Figure 4.
Tandem mass spectrometry (MS/MS) of precursor ion 536.2 m/z using an isolation width of 1.5 m/z and normalized collision energy of 25–35%.
Figure 5.
Extracted pigments and fluorescent compounds of Cryptococcus using large volumes of methanol.
The methanol extracted pigments and fluorescent compounds of C. neoformans var. neoformans (column A), C. gattii, serotype B, (column B) and C. gattii, serotype C (NIH 191, column C). The pink water insoluble pigment (1) was produced by nearly all of the strains tested except for NIH 191, serotype C. The repeated methanol extracts removed most of the brown pigments. NIH 191 was selected because it shows considerable variations from many of the other strains. NIH 191 produced multiple fluorescent compounds that were not produced by most strains of C. gattii. The solvent system was 75% methanol, pH 7.00.
Figure 6.
Extracted pigments and fluorescent compounds of Cryptococcus using small volumes of methanol.
The methanol extracted pigments and fluorescent compounds of C. gattii, serotype B, (198B), C. neoformans var. neoformans (16D), and C. gattii, serotype C (106 and 107). The cells were extracted once with a small quantity of methanol. This type of extraction greatly favored obtaining the fluorescent compounds and minimized the extraction of the brown pigments. The figure also suggests that metabolic differences between the C. gattii strains exist. The solvent system was 75% methanol, pH 7. The molecular mass for several compounds was determined.
Figure 7.
Columns A and B were extracted pigments from C. neoformans var. neoformans; pink pigment 1 was water insoluble and pink pigment 2 was water soluble. Column C shows the extracellular water soluble pink pigment 2 produced by C. neoformans var. grubii. Columns D and E (two dimensional TLC) were the extracted pigments from C. gattii. The solvent was 75% methanol, pH 4 to 5. The acid pH solvent developed the pink intracellular water soluble pink pigment, 2. The molecular weight of both the extracellular or intracellular pink pigment, 2, was 535.2 for each Cryptococcus spp.
Figure 8.
TLC of the pink extracellular pigment.
The TLC results of the C. neoformans concentrated supernatant under visible light, 254 nm, and 365 nm (Section A). The pink pigment separates into a major band (top) and a minor band (lower) which is located at the top of the tryptophan band (Section A). C 18 Sep-Pak column supernatant separation of fluorescent compounds which were then detected on TLC. The fluorescent compounds including tryptophan (blue fluorescence) were eluted with 20% methanol (section B). Multiple 5 ml 20% methanol fraction were collected. After the 8th tube washing with 20% methanol fluorescent compounds were no longer detected in the fractions. The pink pigment was then eluted off the C18 Sep-Pak column with 40% to 50% methanol (section C). The eluted pink pigment formed two bands.
Figure 9.
Laccase mutants of Cryptococcus.
C. neoformans var. neoformans grown on m-LTG agar for two weeks (section I), quadrant a (2ETU-C), quadrant b (B3501), quadrant c (JEC21) and quadrant d (2ETU, laccase mutant). The laccase mutant failed to produce the brown pigment. Similar results were obtained when the same strains were cultured on D-tryptophan (m-FDTG agar) (section II). C. neoformans var. grubii grown on m-FLTG agar for two weeks (section III), quadrant a (H99, lac1 and lac2 positive), quadrant b (MDJ12 (lac1 mutant), quadrant c RPC26 (lac2 mutant), and quadrant d QGC8 (lac1 and lac2 double mutant). When lac1 was deleted, production of the pink pigment was significantly reduced. The TLC illustrated that when the pink pigment was produced intracellularly the laccase mutant of C. neoformans var. neoformans (2ETU) (column d) produced reduced amounts of the pink pigment.