Growth and Pigment Production on d-Tryptophan Medium by Cryptococcus gattii, Cryptococcus neoformans, and Candida albicans▿

Given the increasing prevalence of cryptococcosis caused by Cryptococcus gattii (serotypes B and C) strains, there is a need for rapid and reliable tests that discriminate C. gattii from Cryptococcus neoformans (serotypes A, D, and AD). Seventy-two C. neoformans strains, sixty-seven C. gattii strains, and five Candida albicans strains were analyzed for their ability to grow and produce pigment on minimal d-tryptophan d-proline (m-DTDP) medium, on yeast carbon base d-tryptophan d-proline (YCB-DTDP) medium, and on fructose d-tryptophan glycine (m-FDTG) medium. Of the C. gattii and C. neoformans isolates, 94% and 0% grew on m-DTDP agar, respectively, and 98% and 0% grew in YCB-DTDP medium, respectively. C. gattii produced large amounts of brown intracellular pigment(s) on m-DTDP agar and smaller amounts of yellow-brown (amber) extracellular pigment(s). C. albicans grew on both media and produced a pink photoactivated pigment on m-DTDP agar. C. gattii produced large amounts of brown intracellular pigments on the differential medium m-FDTG, whereas C. neoformans produced smaller amounts of the brown pigments and C. albicans produced a pink pigment. The pigments produced by C. gattii from d-tryptophan were distinct and were not related to melanin formation from 3,4-dihydroxyphenylalanine. Thin-layer chromatography of the methanol-extracted C. gattii cells detected four different pigments, including brown (two types), yellow, and pink-purple compounds. We conclude that tryptophan-derived pigments are not melanins and that growth on m-DTDP or YCB-DTDP agar can be used to rapidly differentiate C. gattii from C. neoformans

Visible and fluorescent microscopy of <i>Cryptococcus.</i>

<p>Visible and fluorescent microscopy (FITC filter) of <i>C. gattii,</i> NIH 112, (top) and <i>C. neoformans</i> var. <i>grubii,</i> H99, (bottom). The majority of the yeast cells have a normal appearance but atypical morphology was occasionally observed after growth on tryptophan. <i>C. gattii</i> and <i>C. neoformans</i> var. <i>grubii</i> cells exhibit strong fluorescence with the FITC filter. <i>C. gattii</i> cells often contained small concentrated foci that were fluorescent. The cell walls of <i>C. neoformans</i> var. <i>grubii</i> were usually fluorescent. The strains of <i>C. neoformans</i> var. <i>neoformans</i> which produced brown pigments exhibited a fluorescent pattern that resembled <i>C. gattii</i> whereas the strains that produced the pink pigment had a fluorescent pattern that was similar to <i>C. neoformans</i> var. <i>grubii.</i></p

TLC of the pink extracellular pigment.

<p>The TLC results of the <i>C. neoformans</i> 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 8^th 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.</p

TLC of pink pigments.

<p>Columns A and B were extracted pigments from <i>C. neoformans</i> var. <i>neoformans</i>; 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 <i>C. neoformans</i> var. <i>grubii.</i> Columns D and E (two dimensional TLC) were the extracted pigments from <i>C. gattii.</i> 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 <i>Cryptococcus</i> spp.</p

Comparison of pigment production from L- and D-Tryptophan by <i>Cryptococcus</i> spp.

a<p>Dark Brown is the color of the pigmentation observed. The dark brown pigment masks pink and yellow intracellular pigments that were also produced and detected by TLC.</p>b<p>A few strains produced both the pink extracellular and brown intracellular pigments.</p>c<p>One strain failed to grow on m-LTG and m-FLTG agar.</p

Pink and brown pigment production by <i>Cryptococcus</i>.

<p>The intensity of the pink and brown pigments were estimated visually. Strong pink pigment was produced in m-LTG media by <i>C.neoformans</i> var. <i>grubii</i> (A) <i>C. gattii</i> (B) formed light brown pigments on quadrants I and IV and <i>C. neoformans</i> var. <i>neoformans</i> and <i>C. neoformans</i> var. <i>grubii</i> 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 <i>C. neoformans</i> var. <i>neoformans</i> produced brown pigments on m-LTG agar (not shown). <i>C. gattii</i> produced an intense brown pigment after growth in m-FLTG medium (C).</p

Pink pigment spectra from 200 to 760 nm.

<p>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.</p

Optimum conditions for the production of the extracellular pink and intracellular brown pigments formed by <i>Cryptococcus</i> spp. from L-tryptophan.

a<p>A scale of 0 to ++++ was used. 0 no pigment formation ++ adequate pigment formation, ++++ excellent pigment formation.</p

Laccase mutants of Cryptococcus.

<p><i>C. neoformans</i> var. <i>neoformans</i> 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). <i>C. neoformans</i> var. <i>grubii</i> grown on m-FLTG agar for two weeks (section III), quadrant a (H99, <i>lac₁</i> and <i>lac₂</i> positive), quadrant b (MDJ12 (<i>lac₁</i> mutant), quadrant c RPC26 (<i>lac₂</i> mutant), and quadrant d QGC8 (<i>lac₁</i> and <i>lac₂</i> double mutant). When <i>lac₁</i> 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 <i>C. neoformans</i> var. <i>neoformans</i> (2ETU) (column d) produced reduced amounts of the pink pigment.</p