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.