Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans

Abstract

The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Delta strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Figure 1. Deletion of CUF1 from C. neoformans yields a copper-sensitive phenotype.

(A) Schematic of the Cuf1 protein from C. neoformans shows a KGRP consensus sequence and multiple cysteine-rich domains (cysteines represented by circles). (B) Phenotypes of CUF1 mutants under various copper conditions. Cells were diluted to an A₆₀₀ of 0.2, serially diluted 1:5, and applied to YPD alone or YPD supplemented with either 750 μM BCS or 10 μM CuCl₂ at 37°C for 3 days. (C) Cells were incubated for melanin production as described in Methods (left panels) or on malt extract for 5 days at 30°C and examined by India ink (middle panels) or incubated on Christensen’s agar for 2 hours (right panels).

Figure 2. Effect of CUF1 deletion on survival in mice and pulmonary fungal burden.

(A) Swiss albino mice were injected by tail vein with 10⁶ of the indicated cells, and their progress was followed until they were moribund. (B) CBA/J mice were intranasally inoculated with 10⁵ of the indicated cells and at the indicated time points (0 weeks corresponds to 24 hours), mice were sacrificed, and their lungs were removed and cultured for growth of fungal cells on YPD. Data are mean ± SEM cfu per organ. n = 3 per time point. Only 1 of the mice infected with cuf1Δ cells and assigned for sacrifice at 3 weeks survived. (C) Swiss albino mice were injected as in A and sacrificed at the indicated time points, after which the indicated organs were removed and cultured for growth of fungal cells on YPD.

Figure 3. CUF1 activates CTR4 under low copper conditions.

(A) Northern blot analysis was performed using RNA from the indicated cells grown on YPD and then incubated for 3 hours in asparagine salts with BCS or copper as indicated. (B) Epifluorescence of C. neoformans expressing a CTR4-GFP fusion protein under the native CTR4 promoter on asparagine salts as in A and supplemented with either 10 μM BCS or 3 μM CuCl₂. BF, bright field. Original magnification, ×1,000. (C) Equivalent cells incubated as in B were analyzed by cell flow cytometry as described in Methods. The shaded histogram corresponds to signal from cells supplemented with Cu, and the open histogram corresponds to signal from cells treated with BCS. (D) Plot of mean peak position of epifluorescence by flow cytometry of CTR4-GFP–expressing cells incubated as in A and supplemented with the indicated concentrations of ether BCS or copper.

Figure 4. Expression of CTR4 in macrophages and mouse brain demonstrates low copper availability during infection.

(A) WT or cuf1Δ cells expressing equivalent CTR4-GFP constructs under the CTR4 promoter were incubated with the macrophage-like cell line J774.16 for the indicated times; 200 intracellular (Intra) and extracellular (Extra) were scored for the presence of epifluorescence at the indicated time points. (B) Representative intracellular fungal cells from A were visualized by epifluorescence and intracellular colocalization by Tetramethylrhodamine-labeled dextran after phagocytosis as described in Methods. Original magnification, ×1,000. (C) C. neoformans cells expressing either CTR4-GFP (open histogram) or empty plasmid (shaded histogram) were injected (10⁶ cells) by tail vein into Swiss albino mice and after sacrifice, fungal cells were harvested from their brains, and epifluorescence was measured by flow cytometry. (D) Representative cryptococcal cells from C were visualized by microscopy after recovery from the brains and lungs of intranasally injected CBA/J mice. DIC, differential interference contrast. Original magnification, ×1,000.

Figure 5. CTR4 expression during human cryptococcosis suggests low available copper stores during fungal infection.

(A) In situ hybridization of C. neoformans–infected brain tissue hybridized with an antisense CTR4 digoxigenin-labeled RNA probe. Arrows indicate alkaline phosphatase–positive yeast cells. (B) In situ hybridization of C. neoformans–infected brain tissue hybridized with a sense CTR4 digoxigenin-labeled RNA probe. Arrows indicate alkaline phosphatase–negative yeast cells. (C) Mucicarmine stain of C. neoformans–infected brain tissue. Arrows indicate mucicarmine-positive yeast cells. (D) H&E stain of C. neoformans–infected brain tissue. Arrows indicate unstained yeast cells. Original magnification, ×400.

Figure 6. CTR4 expression varies among cryptococcal isolates, and high levels of expression confer greater ability to disseminate to the brain in human patients.

(A) Twenty-four C. neoformans primary isolates were obtained from consecutive patients (or an H99 laboratory strain) as described in the Methods. Cells were induced by inoculation in asparagine salts, and RNA was obtained and subjected to Northern blot using a fragment of the indicated gene. Ribosomal RNA (rRNA) was visualized by ethidium bromide staining of gels. (B) Northern blots were conducted in duplicate, and CTR4/ACT1 ratios were calculated for the indicated strains by densitometry. (C) Plot of CTR4/ACT1 ratios according to infected organ. Horizontal lines indicate the mean value for each group. P = 0.04.