3.3 The ability to produce melanin of LAC1knock-down/knock-out strains
Furthermore, to assess the melanin production of LAC1-knockdown/knockout strains, we randomly chose and amplified a knock-down colony of group pS-LAC1-A at 1st passage and a stable albino colony of group PNK-LAC1, comparing them by the Transmission Electron Microscope (TEM) and the laccase activity. With the reference TEM of Emma Camachoet al. ,[36], we could identify that, after grown in DOPA broth for 3 days, WT C. neoformans produced melanin granules of high density and almost spherical shape (Fig. 3A-B). And some of melanin granules had been transported to the cell wall (white arrows in Fig. 3B). However, the cells of group pS-LAC1-A produced much less melanin than the WT cells, and most importantly, the LAC1-knockdown cell hadn’t yet transported the melanin granules to the cell wall after growing in DOPA for 3 days (Fig. 3C-D). The albino mutant cell produced no melanin granules (Fig. 3E-F), but some broadly polydispersed minute spherical L-DOPA particles resulted from auto-polymerization, which existed in the three types of cells (asterisk in Fig. 3). These minute L-DOPA particles were much smaller and had a lower density than melanin granules which can aggregate even up to 1 μm in diameter [36]. Besides, the capsule of LAC1-knockdown cell seemed thinner than that of WT and albino cells by rude evaluation (Fig. 3).
Laccase activity in LAC1-knockdown, albino and WT strains was quantified by incubating in DOPA broth for at least three days. Then the supernatant of all the cultures was quantified spectrophotometrically at 475nm [37]. After three-day incubation, the WT strains exhibited about 3-fold greater laccase activity (p<0.01) than the knock-down strains and about 7-fold greater (p<0.01) than the albino strains (Fig. 4B). When extending the incubation time to five days, the WT and knock-down strains demonstrated melanin accumulation, but the albino mutants did not. The WT strains exhibited more than 4-fold greater (p<0.01) laccase activity than the knock-down strains and about 40-fold greater (p<0.01) than the albino strains (Fig. 4B). Meanwhile, the laccase activity of knock-down strains became about 8-fold stronger (p<0.01) than the albino strains, whereas they were no statistical differences on the 3rd day. This observation was consistent with the phenotype (Fig. 4A) and TEM.