FIG 3.

Virulence-related phenotypes of cryptococcal vps27Δ strains and VPS27 are required for mammalian virulence. (A) Indicated strains were assayed for urease by incubation on Christenson’s media according to Materials and Methods. (B) Indicated strains were assayed for laccase by melanin formation according to Materials and Methods. (C) Indicated strains were assayed for laccase by melanin formation according to Materials and Methods. WT; wild type, EV; empty vector, OE; overexpressor. (D) For quantitative analysis of laccase activity, a colorimetric assay was used as described previously (34), using the laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sufonic acid) according to Materials and Methods. Error bars indicate standard error of the mean (SEM), N = 3 independent experiments. *P < 0.05, ***P < 0.005. (E) Indicated strains were incubated on a 1:10 dilution of Sabouraud (SAB) or RPMI agar for 2 days at 30°C and examined by India ink microscopy. Bar, 5 μm. (F) Results (n > 20 cells) ± SEM, Student’s t test, ****P < 0.001. Wild-type H99, vps27Δ, VPS27 overexpressor, and VPS27 complemented strains were grown to the mid-log phase, resuspended to an OD₆₀₀ of 0.1, and grown in a shaking incubator at (G) 30°C or (H) 37°C. (I) ND40 mice were intravenously infected with 10⁶ wild-type H99, vps27Δ, or vps27Δ + VPS27 complemented strain cells. P < 0.0001.