Cryptococcus neoformans requires the ESCRT protein Vps23 for iron acquisition from heme, for capsule formation, and for virulence

Abstract

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

Fig 1

Growth defect of T-DNA insertion mutants on heme. Tenfold serial dilutions of each strain were spotted on the indicated media, and the plates were incubated at 30°C for 2 days before being photographed. The strains were the parental WT strain H99, the three mutants (272D10, 277A9, and 292E1) with insertions in VPS23, and a mutant with a deletion in the CFO1 gene for the ferroxidase of the high-affinity iron uptake system (10).

Fig 2

Requirement of VPS23 for growth on solid and liquid media with heme as the sole iron source. (A to C) Tenfold serial dilutions of each strain (labeled on the right) were spotted on the indicated media and the plates were incubated at 30°C for 2 days before being photographed. (D) Cells of the WT, the vps23-9 mutant, and the overexpression strain were inoculated into liquid YNB medium plus 150 μM BPS without and with supplementation with iron sources. The cultures were incubated at 30°C, and OD₆₀₀s were measured. (E) The indicated strains were also tested for growth in the defined LIM supplemented with heme or FeCl₃ by the same method used for panel D.

Fig 3

Reduced susceptibility of vps23 mutants to noniron MPs. Tenfold serial dilutions of cells of the indicated strains were spotted onto defined LIM with heme as the iron source. The plates on the right contained GaCl₃, GaPPIX, or MnPPIX. The plates were incubated for 2 days at 30°C. (A, B) The mating type α strains in the parental H99 or the cfo1 mutant background were tested for susceptibility. (C) The vps23 mutant in the mating type a strain (KN99a) background was also tested to confirm the altered susceptibility. Note that 200 μl of the noniron MP IX solutions at the indicated concentrations were spread onto the surface of the plates prior to inoculation.

Fig 4

Defective endocytosis and increased susceptibility to trafficking inhibitors for vps23 mutants. (A) The WT, vps23 mutant, and VPS23-overexpressing strains were grown in YPD medium at 30°C overnight, harvested, washed in PBS (pH 7.2), and stained with FM4-64 on ice for 15 min. The cells were then incubated for an additional 30 min at 30°C before viewing. Bar, 5 μm. DIC, differential interference contrast. (B) Tenfold serial dilutions of the indicated strains were spotted onto YNB alone or YNB supplemented with either 20 μg/ml BFA or 0.5 mg/ml monensin. The plates were incubated for 3 days at 30°C.

Fig 5

Altered capsule size and polysaccharide shedding of vps23 mutants and VPS23-overexpressing strains. (A to C) Cells were cultured in defined LIM at 30°C for 48 h, and capsule formation was assessed by India ink staining of the indicated strains. Bar, 5 μm. (D) Fifty cells of each strain were measured to determine the cell diameter and capsule radius. Each bar represents the average of the 50 measurements with standard deviations. An asterisk indicates that the count is statistically significantly different from the others by the Student t test (P < 0.05). (E) The electrophoretic mobility and quantity of shed polysaccharide were assessed as described in Materials and Methods, by using an anti-GXM antibody to detect the capsule.

Fig 6

Reduced melanization in vps23 mutants. Tenfold serial dilutions of cells of the indicated strains were spotted onto solid l-DOPA plates. The plates were incubated for 2 days at 30°C prior to being photographed.

Fig 7

Loss of Vps23 influences growth at alkaline pH and susceptibility to salt stress. (A) Cells of the indicated strains were grown in buffered YNB, and growth was quantified by monitoring the absorbance at 600 nm after 24 h at 30°C. (B, C) Tenfold serial dilutions of cells of the strains indicated on the right were spotted onto solid YNB without or with 1.5 M NaCl, 1.5 M KCl, 1.5 M sorbitol, or 100 mM LiCl. The plates were incubated at 30°C for the following times: sorbitol, 3 days; KCl, 5 days; NaCl, 5 days; LiCl, 10 days.

Fig 8

Vps23 influences virulence in mice. Ten female BALB/c mice were inoculated intranasally with each of the strains indicated, and the survival of the mice was monitored daily. The mice were each inoculated with 2 × 10⁵ fungal cells. Survival differences between groups of mice were evaluated by log rank tests. The P values for the mice infected with the WT and mutant strains were statistically significantly different (P < 0.001).