Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis

Sun, Tian-Shu; Ju, Xiao; Gao, Hui-Ling; Wang, Tao; Thiele, Dennis J.; Li, Jia-Yi; Wang, Zhan-You; Ding, Chen

doi:10.1038/ncomms6550

Article
Published: 24 November 2014

Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis

Tian-Shu Sun¹^na1,
Xiao Ju¹^na1,
Hui-Ling Gao¹,
Tao Wang¹,
Dennis J. Thiele²,
Jia-Yi Li^1,3,
Zhan-You Wang¹ &
…
Chen Ding¹

Nature Communications volume 5, Article number: 5550 (2014) Cite this article

4524 Accesses
67 Citations
59 Altmetric
Metrics details

Subjects

Abstract

Copper homeostasis is important for virulence of the fungus Cryptococcus neoformans, which can cause lethal meningoencephalitis in humans. Cryptococcus cells encounter high copper levels in the lung, where infection is initiated, and low copper levels in the brain. Here we demonstrate that two Cryptococcus copper transporters, Ctr1 and Ctr4, differentially influence fungal survival during pulmonary infection and the onset of meningoencephalitis. Protein Ctr1 is rapidly degraded under the high-copper conditions found in infected lungs, and its loss has no effect in fungal virulence in mice. By contrast, deleting CTR4 results in a hypervirulent phenotype. Overexpressing either Ctr1 or Ctr4 leads to profound reductions in fungal burden in the lung. However, during the onset of meningoencephalitis, expression of the copper transporters is induced and is critical for Cryptococcus virulence. Our work demonstrates that the fungal cells switch between copper detoxification and acquisition to address different copper stresses in the host.

You have full access to this article via your institution.

Download PDF

A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis

Article 13 January 2020

Fungal kinases and transcription factors regulating brain infection in Cryptococcus neoformans

Article Open access 23 March 2020

Deficiency of lung-specific claudin-18 leads to aggravated infection with Cryptococcus deneoformans through dysregulation of the microenvironment in lungs

Article Open access 26 October 2021

Introduction

Copper (Cu) is an essential metal for life that serves as a critical cofactor for numerous vital biological processes including oxidative stress resistance, energy production, melanin biogenesis and signal transduction^1,2. The activities of proteins such as superoxide dismutase, cytochrome c oxidase, dopamine β-hydroxylase and nitrous-oxide reductase depend on Cu¹. Moreover, numerous studies reveal the critical role of Cu for immunodefense against invading microbial pathogens. Cu-mediated pathogen killing depends on the generation of hydroxyl radicals and hydroxyl anions that causes DNA breakage, protein damage and destruction of iron–sulphur clusters^2,3. Recent studies have extensively demonstrated the function of Cu as an antimicrobial agent. Levels of the mammalian Cu transporters (Ctr1 and ATP7A) in response to interferon-γ or lipopolysaccharides are markedly induced in macrophage cell lines⁴. Alveolar immune cells from mice challenged with Cryptococcus express higher Ctr1 protein levels and accumulate intracellular Cu⁵. Moreover, Cu-ionophores intensify the hostile antimicrobial Cu environment of innate immune cells in coping with C. neoformans infection⁶. As pathogens, bacterial or fungal cells lacking Cu-detoxification machinery including Cu-exporting pumps or metallothioneins (MTs) often exhibit a lower survival rate in the host^4,5,7. Interestingly, recent work demonstrated that the disruption of Aspergillus CtpA Cu transporter causes an increased pathogenicity in the Galleria mellonella infection model⁸.

C. neoformans is one of the most life-threatening human fungal pathogens and is a direct causative agent of lethal meningoencephalitis in both immunodeficient and immunocompetent individuals^9,10. C. neoformans infection initiates in the human lungs, after inhalation of fungal cells from the environment. As the infection progresses, the fungal cells gradually disseminate to the brain, by uncharacterized mechanisms, where meningoencephalitis ensues^9,10,11. Previous studies of C. neoformans virulence factors have implied that Cu plays important, but poorly characterized, roles in fungal virulence. This hypothesis is supported by the Cu dependence of many biological processes and virulence-related proteins, including melanin biogenesis, superoxide dismutase activity and Fe uptake through Cu-dependent ferroxidase activity^12,13,14,15. However, recent studies also demonstrated that the Cu-detoxification proteins known as MTs are essential for fungal survival during pulmonary colonization and for virulence, indicating that C. neoformans encounters elevated Cu levels in the host early in the infectious process⁵.

Here we investigate the role of two functionally redundant, high-affinity Cu⁺ transporters (Ctr1 and Ctr4) in fungal pathogenicity in mice. We demonstrate that loss of CTR1 does not affect fungal survival in the lung because of the normally rapid degradation rate of the Ctr1 protein in a high-Cu environment similar to that found in infected lung tissues. Importantly, loss of Ctr4 results in a hypervirulent phenotype, and overexpression of CTR1 or CTR4 leads to dramatic reductions in colony forming unit (CFU), consistent with C. neoformans cells coping with high Cu levels during the initial phase of infection in the lung. Although ctr1Δctr4Δ cells also exhibit in high CFUs in the lung and brain, they do not have an impact on the survival rate of mice compared with mice infected with wild-type fungal cells. Using an intracerebral infection model to bypass lung tissue, we demonstrate that C. neoformans lacking both Ctr1 and Ctr4 exhibit strongly impaired cell growth in human cerebrospinal fluid (CSF) and reduced virulence in mouse brain. Taken together, these results demonstrate that C. neoformans senses distinct Cu environments in the lung and brain, and that the Cu acquisition machinery plays reciprocal roles in fungal virulence in these distinct niches during the course of infection.

Results

Loss of CTR4 causes hypervirulence in mice

To understand the potential contribution of the Cu-acquisition machinery to C. neofomans virulence, we infected A/J mice intranasally with isogenic H99 (wild-type), ctr1Δ, ctr4Δ or ctr1Δctr4Δ strains (Fig. 1a, Supplementary Fig. 1A and Supplementary Table). No significant differences in survival rates were observed among the mice infected with H99, ctr1Δ or ctr1Δctr4Δ cells. However, ctr4Δ cells exhibited a strong hypervirulent phenotype. Because A/J mice carry a mutation that may be related to lung disease¹⁶, we further validated this result by infecting C57BL/6 mice with H99, ctr4Δ, the CTR4 complementation strain or ctr1Δctr4Δ. In agreement with the results obtained in A/J mice, ctr4Δ cells demonstrated significantly increased virulence, whereas the complementation strain showed reduced virulence compared with H99 cells (Fig. 1b) because of overexpressing CTR4 (discussed below). Similar survival rates were observed for mice infected with ctr1Δctr4Δ cells and mice infected with wild-type cells (Fig. 1c). A fungal burden analysis of the lung and brain tissues was performed in both sets of mice. We detected robust increases in the fungal burden in mice infected for ctr4Δ and ctr1Δctr4Δ, but not for mice infected with ctr1Δ cells (Fig. 1d–g). Because nasal mucosal membranes might capture fungal cells, resulting in uneven inoculation of fungal cells in the lung, an intratracheal infection experiment was also performed. An increased fungal burden was observed in mice infected with ctr4Δ and ctr1Δctr4Δ cells in the lung, consistent with the results for the intranasal route of infection (Fig. 1h).

**Figure 1: Disruption of *CTR4* increases *C. neoformans* virulence in A/J and C57BL/6 mice.**

We also noted that lungs infected with ctr4Δ or ctr1Δctr4Δ were significantly enlarged, compared with those from mice infected with wild-type C. neoformans (Fig. 2a,b), indicating a hyperinflammatory response and greater lung tissue damage. This finding was confirmed by haematoxylin/eosin and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) staining, which showed more infiltration of mononuclear cells and higher numbers of apoptotic lung cells when mice were infected with ctr4Δ or ctr1Δctr4Δ cells as compared with the H99 strain (Fig. 2c–f). This lung tissue enlargement may be due to the production of a more elaborate biofilm structure by ctr4Δ and ctr1Δctr4Δ cells in the lung tissue or on blood vessels. However, no differences in biofilm formation were observed among the H99, ctr1Δ, ctr4Δ or ctr1Δctr4Δ strains (Fig. 2g). We also examined the CFUs in the blood, and we can only detect Cryptococcus cells in the blood, irrespective of genotype, at 21 days post infection (Fig. 2h), as described previously¹⁷. These data strongly demonstrated that the lung tissue enlargement and damage observed at 14 days post infection are due to increased fungal cell proliferation of the ctr4Δ and ctr1Δctr4Δ strains, and not enhanced biofilm formation in the lungs or blood vessels. Given that C. neoformans cells experience high Cu levels in the lung⁵, our results suggest that the loss of CTR4 efficiently reduces host killing of C. neoformans during the initial stages of infection. However, as shown in Fig. 1, while Ctr1 and Ctr4 are independent and functionally redundant integral membrane Cu⁺ importers, the two Cu⁺ transporter mutants exhibit difference in pulmonary infection phenotypes in vivo. In addition, the ctr1Δctr4Δ strain exhibited increased CFUs in the lung and brain CFUs compared with the H99 strain; however, animal survival rates were indistinguishable.

**Figure 2: Disruption of *CTR4* induces inflammatory responses and lung damage.**

Cu transporter expression dictates fungal survival in the host

A deletion of the C. neoformans CTR4 gene was previously generated using an auxotrophic marker (URA5) in an ura5Δ strain that was generated using 5-fluoroorotic acid (5-FOA)¹⁸. The ctr4Δ::URA5 (NIH) (a gift from Dr Peter Williamson, NIH, USA) and ctr4Δ::NEO strains did not exhibit capsule formation¹⁸ or release defects (Supplementary Fig. 1B,C); however, the ctr4Δ::URA5 (NIH) strain did demonstrate a reduction in fungal virulence and defective cell growth under nutrient-limiting conditions¹⁸. To carefully verify our ctr4Δ phenotype (ctr4Δ::NEO), we performed Southern blot analysis of ctr4Δ::NEO and ctr1Δ::NAT ctr4Δ::NEO, which revealed a single integration of the NEO cassette at the CTR4 allele (Supplementary Fig. 1). We also generated a ctr4Δ::URA5 ura5Δ::HYG strain (Supplementary Table) and confirmed the lack of CTR4 mRNA expression with real-time PCR (Fig. 3b). No differences in growth defects were observed between the wild-type strain and the isogenic Cu⁺ transporter mutants under nutrient-deficient conditions, regardless of the addition of Cu, bathocuproine sulphonate (BCS) or uracil (Supplementary Fig. 2A). Although we validated the correct integration of the CTR4::URA5 cassette in our ctr4Δ::URA5 strain with integration-diagnostic PCR, we were unable to confirm correct integration in ctr4Δ::URA5 (NIH) cells (Supplementary Fig. 3A,B). The loss of the wild-type CTR4 allele was detected in both ctr4Δ::URA5 strains. Interestingly, we could not detect an intact ctr4Δ::URA5 allele in the CTR4 complementation strain (NIH), which should maintain URA5 at the CTR4 allele and a wild-type randomly reintegrated CTR4 allele, as mentioned previously¹⁸ (Supplementary Fig. 3A). Southern blot analysis (Supplementary Fig. 3C) confirmed the presence of a wild-type URA5 in H99, ctr4 Δ::URA5 (NIH) and the CTR4 complementation strain (NIH), and the integration of URA5 at the CTR4 allele in the ctr4Δ::URA5 strain (NIH). However, the complementation strain does not possess a ctr4Δ::URA5. We next determined whether the nutrient-dependent phenotype of ctr4Δ::URA5 (NIH) is because of uracil. The cell growth of ura5Δ and ctr4Δ::URA5 (NIH) was significantly inhibited when uracil was absent in the media (Supplementary Fig. 4A). The expression of URA5 was significantly induced in ctr4Δ::URA5 (NIH) cells in the absence of uracil, perhaps indicating that uracil starvation induces native nonfunctional URA5 in the 5′ FOA-mutagenized strain. Because we were uncertain by the genotype of the CTR4 complementation strain (NIH), we transformed a GPD promoter-CTR4HA construct into ctr4Δ::URA5 (NIH) and ctr1Δctr4Δ cells using the biolistic transformation method. CTR4HA protein expression was then examined in the resulting ctr4Δ::URA5 (NIH) GPD-CTR4HA transformant. Our data demonstrated that CTR4HA expression can complement ctr1Δctr4Δ cell growth under Cu deficiency, while ctr4Δ::URA5 (NIH) cells expressing CTR4HA retained the uracil-dependent phenotype (Supplementary Fig. 4B). Therefore, these analyses demonstrate that the previously used CTR4 complementation strain phenotype is not reproducible in our hands and suggest that CTR4 is not involved in general nutrient acquisition in C. neoformans (Supplementary Figs 3 and 4)¹⁸. As our ctr4Δ::URA5 strain exhibited a trend of reduced URA5 expression and the ctr4Δ::URA5 (NIH) cells incorrectly demonstrated the CTR4 phenotype, both strains were excluded from the animal infection experiments to avoid complications in the interpretations of the results.

**Figure 3: Overexpression of *C. neoformans* Cu transporters impairs fungal survival in intranasal infections.**

As shown in Fig. 1, the CTR4 complementation strain was less virulent than the isogenic H99 strain. Quantitative reverse transcription–PCR (qRT–PCR) analysis revealed that expression of CTR4 in the complemented strain was higher than that in the wild-type; therefore, we hypothesised that the reduced virulence phenotype was because of the overexpression of CTR4. To test this hypothesis, we overexpressed CTR1 or CTR4 in the H99 or ctr1Δctr4Δ strains, respectively. All strains overexpressing the Cu transporters were used for fungal burden analysis (Fig. 3a,b). We showed that all strains with higher expression of either the Ctr1 or Ctr4 Cu transporter had significantly reduced fungal burdens in lung and brain tissues (Fig. 3c–f).

Previous inductively coupled plasma mass spectrometry analysis suggested that the ctr4Δ::URA5 (NIH) strain exhibited higher cell-associated Cu levels than wild-type cells, implying that the ctr4Δ::URA5 (NIH) strain has more bioavailable intracellular Cu¹⁸. However, the inductively coupled plasma mass spectrometry (ICP-MS) method quantifies Cu from whole cells and does not necessarily reflect intracellular Cu status. Moreover, the pleiotropic growth phenotype observed for the ctr4Δ::URA5 (NIH) strain is independent of CTR4 (Supplementary Figs 3 and 4)¹⁸. To determine the contribution of the C. neoformans Cu⁺ transporters to intracellular Cu availability, we assayed the activity of Cu/Zn-superoxide dismutase (SOD) in wild-type and Cu⁺ transporter mutant strains because Cu/Zn-SOD activity depends on the amount of bioavailable intracellular Cu¹⁹. Although the Cu/Zn-SOD activities of the H99, ctr1Δ and ctr4Δ strains were similar, that of the ctr1Δctr4Δ strain was significantly reduced by 40% in SC medium and >50% in the medium that was Cu-limited by addition of the extracellular Cu⁺ chelator, BCS (Fig. 3g), indicating that CTR1 and CTR4 are functionally independent Cu⁺ importers and that the disruption of either Cu transporter does not significantly influence intracellular Cu status.

CTR4 has been shown to negatively regulate the expression of C. neoformans MTs; however, it is not clear whether the pyrimidine biosynthesis pathway, or the Ctr4 Cu⁺ transporter, influences expression of MTs¹⁸. In our hands, expression of both CMT1 and CMT2 are significantly reduced in the absence of CTR4 when cells encountered high-Cu conditions and in the lungs (Fig. 4a,b), consistent with CTR4 encoding a high-affinity Cu⁺ importer in C. neoformans. To determine whether the hypervirulent phenotype of ctr4Δ is due to the modulation of expression of CMTs, as shown previously¹⁸, we deleted CTR4 in the cmt1Δcmt2Δ strain (Supplementary Table and Fig. 4c,d). The cmt1Δcmt2Δctr4Δ strain did not exhibit a nutrient-dependent phenotype (Supplementary Fig. 2B). Intranasal infection and the fungal burden analysis revealed that fungal colonization was reduced in the lungs and brains of mice infected with cmt1Δcmt2Δ (Fig. 4c,d). Deleting CTR4 in the cmt1Δcmt2Δ strain rescued the survival of cmt1Δcmt2Δ cells in the lung and brain to near wild-type levels (Fig. 4c,d), demonstrating that Ctr4 independently regulates fungal virulence, regardless of the presence of MTs.

**Figure 4: Ctr4 modulates fungal virulence independently of the regulation of metallothioneins.**

The Ctr1 and Ctr4 proteins have distinct properties

Our data demonstrated that the ctr1Δ strain does not exhibit changes in fungal virulence but that strains overexpressing CTR1 results in decreased fungal burden. One possible explanation is that Ctr1 is rapidly degraded in the presence of high Cu levels. We measured the Ctr1 protein half-life under Cu-depleted conditions. To eliminate the effect of the overexpression system on the protein-level quantification, we generated integration cassettes that insert a DNA sequence encoding a HA tag before the stop codon in the genes of interests to compare the proteins expressed from their native loci (Fig. 5a). We first repressed or induced protein expression by culturing cells in the presence of Cu or BCS, respectively, and robust protein expression was detected for both the Ctr1 and Ctr4 proteins under low-Cu conditions and for Cmt1 under high-Cu conditions (Fig. 5b and Supplementary Fig. 5A). We then extensively induced the expression of CTR1HA and CTR4HA in the presence of 1 mM BCS for 6 h and measured the Ctr1HA and Ctr4HA protein stability in response to high Cu levels. As shown in Fig. 5c and Supplementary Fig. 5B, the Ctr1HA protein was not detected after a 5-min exposure to Cu, whereas Ctr4HA was maintained for at least 90 min after Cu addition. These data strongly suggest that, in contrast to Ctr4, the Ctr1 protein is highly unstable under high-Cu conditions. To ascertain whether Ctr1 is expressed in the lung, we performed immunohistochemistry. Although expression of Cmt1HA and Ctr4HA was detected 2 days post infection, we detected little to no signal for Ctr1HA (Fig. 5d).

**Figure 5: *C. neoformans* Cu transporters have distinct properties.**

Interestingly, we detected two protein species for both Cu transporters (Fig. 5b, c, Supplementary Fig. 5A,B), suggesting that these Cu⁺ transporters undergo post-translational modification. In mammals, the Ctr1 Cu⁺ transporter is glycosylated at two positions within the N terminus^20,21,22,23. We used a protein deglycosylation enzyme mix that removes both N- and O-glycosylation to determine whether C. neoformans Cu transporters are glycosylated. After incubation, we were unable to detect the upper band for Ctr4HA, suggesting that Ctr4 is glycosylated. Experimental results under identical conditions gave no evidence that Ctr1 is glycosylated (Fig. 5e and Supplementary Fig. 5C). The prediction algorithms from the NetNGlyc and NetOGlyc web servers showed that the Asn19 residue on Ctr4 is a potential N-glycosylation site. A candidate glycosylation-resistant mutant was generated by integrating a mutated DNA sequence at the CTR4-native allele (Supplementary Fig. 6A). Correct integration was confirmed with PCR (Supplementary Fig. 6A) and gene copy tests with real-time PCR (Supplementary Fig. 6B). The immunoblot analysis demonstrated that mutating Asn19 completely abolishesd the glycosylation of Ctr4HA (Fig. 5f and Supplementary Fig. 5D). Furthermore, the glycosylation mutant rescued the ctr1Δctr4Δ growth phenotype in the presence of BCS, although not to the level of the wild-type CTR4 complementation strain (Fig. 5g), suggesting that fully functional Ctr4 requires glycosylation. As protein glycosylation is processed on endoplasmic reticulum (ER) and may affect protein trafficking, we performed indirect immunofluorescence assays and observed plasma membrane and ER-associated protein localization for both the wild-type Ctr4 and N19Q-mutant proteins, suggesting that Ctr4 trafficking is not dependent on N19 glycosylation (Supplementary Fig. 6C).

Ctr1 and Ctr4 are critical during meningoencephalitis

Although the ctr1Δctr4Δ-mutant strain exhibited higher fungal burden in the lung and brain, surprisingly, identical survival was observed in mice infected with ctr1Δctr4Δ or H99 cells (Fig. 1a). We hypothesized that ctr1Δctr4Δ cells counter Cu toxicity in the lung, leading to high fungal intrusion into the brain, where Cu transporters are required for virulence. To test this hypothesis, we employed ex vivo two-photon microscopy analysis of lung and brain tissues to examine the fluorescence intensity of green fluorescent protein (GFP) that is driven by either the CTR4 or CMT1 promoters. One and seven days post intranasal or intracerebral infection, the CMT1p-GFP produced higher GFP signals in the lung than that of CTR1p-GFP and CTR4p-GFP (Fig. 6a), as previously described⁵. In the brain, the fluorescence intensities of CTR1p-GFP and CTR4p-GFP were higher than that of CMT1p-GFP (Fig. 6b). These data strongly suggest that C. neoformans cells sense Cu-limitation conditions in the host brain. In human CSF, the copper level is ~7.5±3.1 ng ml⁻¹ (~0.12±0.048 nmol ml⁻¹)²⁴. As access to the host CSF is critical for cryptococcosis, we examined fungal cell growth in human CSF with or without the supplementation of Cu. CFU analysis indicated that the growth of the C. neoformans Cu-acquisition mutant (ctr1Δctr4Δ) was strongly limited in the absence of exogenous Cu (Fig. 6c). Interestingly, the addition of Cu also stimulated the growth of wild-type cells after 96-h incubation, indicating that Cu is important for C. neoformans to survival in the CSF.

**Figure 6: *C. neoformans* senses Cu deficiency in the host brain and human CSF.**

To bypass the lung, mice were infected intracerebrally with H99, ctr1Δ, ctr4Δ and ctr1Δctr4Δ strains. Mice infected with H99, ctr1Δ and ctr4Δ exhibited similar survival rates, while the ctr1Δctr4Δ strain exhibited a significant reduction in fungal virulence (Fig. 7a), indicating that either of the functionally redundant Ctr1 or Ctr4 Cu⁺ importers is required for C. neoformans survival in the brain. To further address the Cu environment that is sensed by C. neoformans in brain tissue, we injected mice with H99, cmt1Δcmt2Δ or ctr1Δctr4Δ and demonstrated that MTs play no role in C. neoformans survival in the brain (Fig. 7b). These data suggest that C. neoformans cells do not experience a Cu toxicity environment, but rather a Cu-deficient environment in the brain that limits virulence. When a higher fungal cell inoculum was used for intracerebral infection of ctr1Δctr4Δ, increased fungal virulence was observed (compare Fig. 7b,c), which may explain the difference in the survival rates of mice infected with H99 and ctr1Δctr4Δ in Fig. 1a. Given that intranasal infection with ctr1Δctr4Δ produced a fivefold increase in CFUs in the brain compared with infection with H99 (Fig. 1), these observations support the hypothesis that the low-Cu conditions of the brain inhibit fungal cell proliferation of ctr1Δctr4Δ, resulting in similar animal death rates. It has been shown that in response to interferon (IFN)-γ activation, macrophages robustly express the mammalian Ctr1 Cu⁺ transporter and accumulate antimicrobial Cu in the phagosomal compartment^4,25. To further analyse immune responses in the lung and brain, we measured the production of two important cytokines (IFN-γ and tumour necrosis factor (TNF-α) after 7 or 14 days in uninfected mice and mice intranasally infected with C. neoformans. We show here that IFN-γ and TNF-α were significantly increased in response to C. neoformans infection in the lung, whereas their levels remained constant in the brain (Fig. 7d).

**Figure 7: *C. neoformans* Cu⁺ transporters are required for meningoencephalitis.**

Discussion

Human pathogenic fungi adapt to distinct environments by rapidly altering gene expression in response to environmental stimuli. To survive, replicate and invade host tissues, pathogenic fungi have evolved efficient and sophisticated machineries to compete for essential nutrients, including metals. In the last decade, studies have implied that Cu is a key virulence modulator for C. neoformans and is required for fungal virulence^{13,14,18,26,27,28}. Fungal pathogens require Cu for maintaining normal cell growth and are equipped with Cu-detoxification defences, suggesting the possibility that fungal cells encounter both Cu scarcity and toxicity during their infectious life cycles. The regulation of Cu acquisition and detoxification is mediated by the transcription factor, Cuf1. The first report of the regulation of Cu homeostasis by Cuf1, published by in ref. 29, revealed that a cuf1Δ strain is sensitive to both low and high Cu^12,29. A subsequent study showed that Cuf1 controls the expression of the Cu transporter CTR4 and that the cuf1Δ strain is avirulent in a tail vein infection model, thus suggesting that Cu acquisition is essential for C. neoformans virulence in mice^18,27. However, recent work has demonstrated that Cuf1 activates both the Cu-detoxification machinery and the Cu-acquisition machinery, in response to high or low Cu conditions, respectively¹². Moreover, although C. neoformans senses high Cu in the lung and Cu-detoxification genes are critical for fungal survival in the lung, CTR4 is still expressed in the lung at relatively low levels^5,18,30.

What roles does C. neoformans Cu homeostasis play during fungal infection? C. neoformans possesses two high-affinity Cu⁺ importers (Ctr1 and Ctr4) belonging to the Ctr family, which is conserved from yeast to mammals¹². The Ctr family forms homotrimer structures to generate a Cu⁺-importing channel^1,31. Ctr1 and Ctr4 are independent and functionally redundant Cu⁺ importers whose expression is regulated by Cu^12,32. The recently published nutrient-dependent phenotype of the C. neoformans ctr4Δ mutant is of great interest but unexpected¹⁸, and this phenotype has never been observed in other organisms, including Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Aspergillus fumigatus, Drosophila and mice^{8,33,34,35,36,37}. Interestingly, the nutrient phenotype of CTR4 in C. neoformans has not been reported in other studies. For example, two independent studies demonstrated robust cell growth of cuf1Δ strains under nutrient-deficient condition^12,32, given Cuf1 is a positive regulator of CTR4 (ref. 12, 27). The nutrient phenotype of the ctr4Δ strain has not been reproduced while generating the ctr4Δ mutant using other markers, such as NEO, HYG or URA5 (Supplementary Table, Figs 1 and 4, Supplementary Figs 2 and 3)¹². Generating mutants using the URA cassette and a parent strain obtained from 5-FOA mutagenesis are not optimal because previous reports have demonstrated that the ura mutant affects fungal virulence, colony morphology and biofilm formation^{38,39,40,41,42}. The reintegration of wild-type URA at other chromosomal loci does not fully reverse the phenotype and activity of orotidine 5-phosphate decarboxylase^39,40,41,42. In our hands, the ctr4Δ::URA5 (NIH) strain exhibited a uracil-dependent phenotype. Moreover, expressing GPD-CTR4HA in this strain did not complement the phenotype (Supplementary Fig. 4).

Because C. neoformans senses elevated Cu levels in the lung, Ctr4 negatively regulates C. neoformans virulence during pulmonary infection, leading to a remarkable induction of fungal growth, hyperinflammatory responses and severe lung cell damage (Figs 1 and 2). The greater fitness of ctr4Δ and ctr1Δctr4Δ cells in lung tissue results in the increased dissemination of fungal cells into brain tissue. Furthermore, overexpressing the Ctr1 or Ctr4 Cu transporters greatly enhances fungal killing by pulmonary antimicrobial Cu, strongly indicating that Cu transporters negatively influence fungal virulence in the lung (Fig. 3). A Ctr4 mutant has been shown to accumulate Cu and affect MT expression¹⁸, although it is unclear whether the induction of MTs is because of pyrimidine biosynthesis or Ctr4. In S. cerevisiae, the disruption of Cu importers reduces the activation of the MT promoter under high-Cu conditions⁴³. In the present study, deleting C. neoformans CTR4 reduces the expression of MTs both in vitro and vivo, demonstrating that deleting the high-affinity Cu⁺ importer CTR4 reduces the activation of Cu-detoxification machinery (Fig. 4a,b). Irrespective of how CTR4 modulates the expression of MTs, the hypervirulent phenotype of ctr4Δ is not because of the induction of MTs (Fig. 4c,d).

In the present work, differing levels of protein stability and post-translational modification of Ctr1 and Ctr4 were observed. While ctr1Δ does not affect animal survival or CFUs relative to the wild-type parental strain (Fig. 1), Ctr1 is extremely unstable in the presence of exogenous Cu and in the lung (Fig. 5c,d). Interestingly, a strong candidate for an N-glycosylation site was detected in Ctr4 near the putative amino-terminal extracellular domain Cu-binding motifs. The glycosylation of Ctr4 resembles that of mammalian Ctr1, in which glycosylation influences Cu uptake²⁰. Although the N19Q mutant restored Cu uptake in ctr1Δctr4Δ cells, complemented cells exhibited reduced growth compared with ctr1Δctr4Δ cells expressing wild-type CTR4, implying that N-glycosylation is involved in facilitating Ctr4 function in an undefined manner.

Ctr4 has been hypothesized to be important for cryptococcal meningoencephalitis^18,27. Our results provide the first evidence that C. neoformans switches from Cu detoxification to acquisition modes when it disseminates from the lung to the brain (Fig. 8a). The increased fitness of ctr4Δ and ctr1Δctr4Δ cells in the lung leads to a higher fungal proliferation, which, in turns, increases infiltration of fungal cells into the brain, where C. neoformans requires Cu (Fig. 8b). Because the ctr4Δ strain contains a functional Ctr1 Cu⁺ transporter, ctr4Δ cells are able to efficiently acquire Cu from the brain, leading to a hypervirulent phenotype (Fig. 8b). Although the number of ctr1Δctr4Δ cells in the brain is increased compared with the wild-type H99, the Cu-deficient environment in the brain does not permit the robust growth of ctr1Δctr4Δ cells (Fig. 8b). Interestingly, infection routes determine the virulence phenotype of ctr1Δctr4Δ, implying that entry rather than replication of C. neoformans in the brain is critical for fungal virulence in the mouse model^44,45. Because the ctr1Δctr4Δ strain remains virulent in the intracerebral infection model, we speculate that there are other Cu uptake machineries in C. neoformans that are active in Cu transport during the formation of meningoencephalitis.

**Figure 8: A model of the function of the *C. neoformans* Cu homeostasis machinery during systemic infection.**

Little is known about cryptococcal meningoencephalitis and brain immunity. In fact, the brain is a site of immune privilege, with unique immune responses compared with other tissues⁴⁶. Brain immune cells must maintain a balance between killing invading microbes and preventing robust inflammatory responses to protect fragile neurons⁴⁶. Despite the importance of microglial cells in protection against C. neoformans, the expression of brain proinflammatory cytokines (TNF-α, Interleukin (IL)-12 and IL-1) is low at the early stages of infection⁴⁷. Furthermore, damage to the central nervous system causes inhibition of NF-κB in astrocytes, which, in turn, blocks the expression of a range of cytokines, including mammalian Cu transporter inducers, such as IFN-γ⁴⁸. Our data demonstrated that the production of cytokines, including IFN-γ and TNF-α, is induced by C. neoformans infection in the lung tissues but not in the brain. Most importantly, Cu accumulation is often associated with neurodegenerative disease⁴⁹. In conclusion, our study highlights the reciprocal roles of C. neoformans Cu transporters in the host, and underscores the importance of analysing host niches during the investigation of Cryptococcus pathogenicity.

Methods

Strains and media

The C. neoformans H99 strain was generously provided by Ying-Lien Chen (National Taiwan University). The ctr4Δ::URA5 strain was a gift from Dr Peter Williamson (National Institute Health, USA) to Dr Jia-Yi Li (Lund University, Sweden). All mutants (Supplementary Table) were routinely grown in YPD (1% yeast extract, 1% peptone and 2% dextrose) or synthetic complete (SC: 0.67% yeast nitrogen base, 2% dextrose and 0.2% amino acids) as previously described and generated as described below⁵. SC (114400022) and SC-ura dropout mixes (114410622) were purchased from MP Biomedicals. For biolistic transformation, YPD 2% agar supplemented with 1 mM sorbitol was used. For selection of transformants, nourseothricin or G418 was added to YPD at a final concentration of 100 μg ml⁻¹, and hygromycin B was added to YPD at a final concentration of 200 units ml⁻¹.

Generation of Cryptococcus mutants

CTR1 and CTR4 overexpression strains were generated as described below. The genomic DNA from CTR1 and CTR4 were amplified using primers (5′-GGGAACAAAAGCTGGAGCTCCCTTTGCGGAAGTAAAGAAGC-3′ and 5′-ATCCACTAGTTCTAGATCGAATGGCCAGTAAGGAACT-3′ for CTR1, and 5′-GGGAACAAAAGCTGGAGCTCTTTGGTCGACAGAACAATCGG-3′ and 5′-ATCCACTAGTTCTAGAACATCCCTCGTCTCATCCAC-3′ for CTR4. PCR products were cloned into the pHYG-7KB plasmid⁵⁰, between the SacI and XbaI sites using the In-Fusion PCR cloning kit (Clontech). The cloned plasmids were used to transform C. neoformans strains by biolistic transformation⁵¹. Strains were isolated, and the RNA expression levels of CTR1 and CTR4 were confirmed using real-time PCR.

The ura5Δ strain was generated by homologous recombination method. Primers 5′-NNNNCTCGAGTAGTCCTCAGTTTTTGATTCTACTTG-3′ and 5′-NNNNCCCGGGGATCAAGTTCTTATTTGGTCGCCGTTG-3′ were used to amplify the upstream sequence, and primers 5′-NNNNGGATCCAGGGTTTTCTTGGATGCAGGGGT-3′ and NNNNGAGCTCTCTCTCTCTCCCAAACCAAAGTCTC-3′ were used to amplify the downstream sequence. The PCR products were cloned into the pHYG plasmid (containing a hygromycin resistance marker under the control of the Cryptococcus ACT1 promoter), the upstream sequence was inserted between the XhoI and XmaI sites and the downstream sequence was inserted between the BamHI and SacI sites. The cloned plasmid was amplified using the primers 5′-NNNNCTCGAGTAGTCCTCAGTTTTTGATTCTACTTG-3′ and 5′-NNNNGAGCTCTCTCTCTCTCCCAAACCAAAGTCTC-3′ to generate a knockout cassette. The PCR product was then used to transform the wild-type H99 strain. Transformants were first tested for growth in the absence of uracil, and the integration of the knockout cassette was confirmed with Southern blott analysis. The ctr4Δ::URA5 strain was generated exactly as described previously¹⁸. The cmt1Δcmt2Δctr4Δ strain was generated as described previously¹², except the hygromycin resistance marker was used.

The HA integration cassette (pHA-NEO) was generated as described below. The primers 5′-NNNNCCCGGGTACCCATACGATGTTCCAGATTACGCTTAAGGGTTACAGTAGCAGTAGTAATG-3′ and 5′-CTCCAGCTCACATCCTCGCAGCTCTTCACGTGGACGCTCCAAGG-3′ were used to amplify the terminator sequence from GPD1, and the primers 5′-GCTGCGAGGATGTGAGCTGGAG-3′ and 5′-NNNNGGATCCTTTAGGCCTAAGAGATGTAGAAACTAGCTTC-3′ were used to amplify the neomycin resistance marker. The two PCR products were then joined by overlapping PCR, and the resulting product was cloned into the pBluescript plasmid between the XmaI and BamHI sites. For genomic integration of an HA tag at the 3′ end of the gene of interests, the stop codons of CTR1, CTR4 and CMT1 were deleted and replaced with the HA tag-TAA-GPD terminator-HYG marker construct (Fig. 5a). Briefly, downstream sequences (1 kb after the stop codon) were amplified using the primers 5′-NNGGATCCGGGGTGCGTATGTATGCATGT-3′ and 5′-NNTCTAGATCGAATGGCCAGTAAGGAACT-3′ for CTR1, 5′-NNGGATCCAAGAGTAATAATATGTACCGGATGTTGATAG-3′ and 5′-NNTCTAGAAACATTACCTAGGATTCCCTATCCTCT-3′ for CTR4, and 5′-NNGGATCCGCTTGTCTTCCAGACCAATG-3′ and 5′-NNGAGCTCCTTTTGAGAATGCCGAGAGG-3′ for CMT1. The PCR products were then cloned into the pHA-NEO plasmid between the BamHI and XbaI sites for CTR1 and CTR4, and the BamHI and SacI sites for CMT1. Upstream sequences of CTR1, CTR4 and CMT1 were amplified using the primers 5′-NNGGGCCCTGTCCTCTACATCCTCCAACA-3′ and 5′-ATGCCCAACATGACTTGACCC-3′ for CTR1, 5′-NNGGGCCCGACATGGGTAACATGGGCAT-3′ and 5′-ACAACAGCGCCCCTGAGCAC-3′ for CTR4, and 5′-NNCTCGAGAGAACACATCTGCATCCGATA-3′ and 5′-GGCAGCGCCAGATCCGCTGC-3′ for CMT1. The PCR products were then cloned into the pHA-NEO plasmid, between the ApaI and SmaI sites for CTR1 and CTR4, and the XhoI and SmaI sites for CMT1. The integration cassettes were then amplified, and the resulting PCR products were used to transform the wild-type H99 strain. Transformants were tested by diagnostic PCR, and correct integrations of the HA tag were confirmed with western blot analysis.

The CTR4 open reading frame was mutated by PCR (Supplementary Fig. 6A). CTR4 genomic DNA was amplified using two primer pairs 5′-NNNNCTCGAGTTGTTTTGGTCCGTTGA-3′ and 5′-CATGTGGCTGTGCTGGTGGCCGGAGTCCATACCCATGCCCATGCC-3′, and 5′-CCGGCCACCAGCACAGCCACATG-3′ and 5′-NNNNCCCGGGTTAAGCGTAATCTGGAACATCGTATGGGTAACAACAGCGCCCCTGAGCAC-3′. The resulting two PCR products were joined by overlapping PCR, and the resulting product was clond into the CTR4HA integration cassette, to replace the CTR4 upstream sequence. The cloned plasmid was sequenced to confirm the mutated codon. The correct mutation plasmids were then amplified to generate integration cassettes, which were used to transform ctr1Δctr4Δ cells. Correct integrations were confirmed by diagnostic PCR, followed by spotting assays and western blots.

Capsule formation and release

Cells were grown overnight in SC medium and then washed three times with H₂O. The cells were resuspended in RPMI medium supplemented with 10% fetal calf serum (FCS) and incubated for 3 days at 37 °C, 5% CO₂. Capsule release experiments were performed as previously described⁵². Briefly, capsule release was induced in RPMI medium supplemented with 10% FCS for 5 days at 37 °C, 5% CO₂. One millilitre aliquot of cell culture was removed and incubated at 70 °C to denature enzymes. The resulting supernatant samples were filtered using a 0.2-μm filter to remove fungal cells, followed by separation in an agarose gel at 4 °C and 25 V overnight. The capsule molecules were then transferred on a nylon membrane. Immunoblotting was performed using the monoclonal antibody 18B7 at a 1:1,000 dilution⁵³.

Biofilm formation

Cryptococcus cells were grown in SC medium overnight at 30 °C. The cells were then washed three times with PBS and resuspended to an A₆₀₀ of 1.0 in SC medium. The cell culture (100 μl) was transferred to 96-well plates, and the plates were incubated at 37 °C for 24 h. Each well was washed with PBS five times to remove non-adherent cells. A 100-μl aliquot of 0.1% crystal violet solution was added into each well and incubated for 15 min at 30 °C. Excess crystal violet was washed five times with PBS. A 100-μl aliquot of 30% glacial acetic acid was added and incubated for 4 h to release the crystal violet dye from the biofilm structure. Biofilm production was then quantified by measuring the absorbance of the released crystal violet dye at 570 nm.

Nucleic acid isolation and real-time PCR

DNA was isolated as previously described³⁸. Briefly, 5 ml of an overnight Cryptococcus cell culture in YPD was washed with water and resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 1% SDS, 2% Triton-X). Cells were lysed using a bead beater. The supernatant was mixed with 275 μl of 7 M ammonium acetate for 5 min at 65 °C. The DNA was precipitated with isopropanol, washed with 70% ethanol and resuspended in 50 μl of H₂O supplemented with RNase A. One microgram of genomic DNA was used for PCR, and 20 μg of genomic DNA was used for Southern blot analysis. RNA was isolated using the TRIzol reagent (Life Technologies), and Turbo DNase I (Life Technologies) was used to eliminate genomic DNA contamination. One microgram of total RNA was used for cDNA synthesis, using the GoScript Reverse Transcription System (A5001, Promega), and real-time PCR was performed using a CFX Connect Thermocycler (Bio-rad) as previously described⁵. RNA was isolated from mice infected with C. neoformans as previously described³⁰. Briefly, infected lung tissues were first cut into small pieces and resuspended in TRIzol reagent. Glass beads were added, and the mixture was processed using a bead beater. The gene copy number of Ctr4 glycosylation mutants was characterized using real-time PCR as previously described⁵⁴.

Animal and human CSF CFU experiments

All animal experiments were reviewed and ethically approved by the Research Ethics Committees of the College of Life and Health Sciences of Northeastern University. Six- to eight-week-old female A/J and C57BL/6 mice were purchased from the National Cancer Institute (USA) and Changsheng Biotech (China), respectively, and used for infection and fungal burden assays. Intranasal and intracerebral infections were performed as previously described^5,55. For intranasal infections, mice were anaesthetized and intranasally infected with 10⁵ fungal cells in 50 μl PBS. For intracerebral infections, mice were anaesthetized and Cryptococcus cells in 3 μl of PBS were injected into lateral ventricle using a mouse brain stereotactic instrument. Fungal burden assays were performed as described below. Infected mice were killed by exposure to CO₂ for 5 min. Lung and brain tissues were isolated, weighed and placed in 5 ml of PBS buffer. The tissues were then homogenized, and the suspension was plated on SC agar at 30 °C for 3 days. For immunohistochemistry, tissues were isolated and fixed in paraformaldehyde. Fixed tissues were frozen and tissues sections (10-μm thick) were processed using a Crypstat Microtome Leica CM1850 (Leica). Immunohistochemistry and haematoxylin and eosine staining were performed as previously described⁵. TUNEL staining was performed according to the manufacturer’s instructions (Roche). The human CSF sample was kindly provided by Dr Hao Zhang, MD (China Medical University). The CSF samples were pooled from three patients, each with a written formal consent. The utilization of human CSF samples was reviewed and ethically approved by the Laboratory Ethics Committees of China Medical University and the Research Ethics Committees of the College of Life and Health Sciences of Northeastern University. The CFU in human CSF was performed as described previously⁵⁶.

Protein quantifications and SOD activity measurements

Immunoblotting signals were captured using ChemiDoc XRS+ (Bio-Rad). Anti-HA (ab9110, Abcam; 1:1,000 dilution) and anti-histone H3 antibodies (4499P, Cell Signaling; 1:1,000 dilution) were used. The Protein Deglycosylation Mix kit (P6039S, NEB) was used as described in the manufacturer’s manual. Briefly, an overnight culture of fungal cells were subcultured in SC medium supplemented with 1 mM BCS for 6 h at 30 °C. The cells were resuspended in HEPES buffer (50 mM HEPES, 140 mM NaOH, 1% Tritone X-100, 1 mM EDTA and protease inhibitors). Proteins were isolated using a bead beater and incubated with the deglycosylation enzyme mixture for 4 h at 37 °C under denaturing conditions or nondenaturung condition according to the manual of Protein Deglycosylation Enzyme kit (NEB). The reaction mixtures were then separated by 15% SDS–PAGE. Immunoblottings were then performed using the HA antibody. Mouse IFN-γ (ELM-IFNg) and TNF-α (ELM-TNFa) ELISA kits were purchased from RayBiotech, and experiments were performed according to the manufacturer’s instructions. The SOD activity was quantified using the SOD activity kit (Jiancheng Bio) according to the manufacturer’s instructions.

Cryptococcus indirect immunofluorescent microscopy

The immunofluorescent microscopy was performed as described previously¹². Cells were grown in SC medium overnight and then subcultured for additional 3 h in the presence of 100 μM BCS to induce protein expression levels of Ctr4HA and N19Q mutant. Formaldehyde was then added to fix the cells, and the cells were incubated at 4 °C for 1 h with gentle shaking. The cells were then washed with PBS and resuspended in sorbitol buffer. Lysing enzyme (L1214, Sigma) supplemented with protease inhibitor (Roche) was used to digest the cell wall at 37 °C. Cells were then washed with PBS and resuspended in a PBS BSA solution and adhered to a glass slide treated with poly-lysine. The anti-HA antibody (1:300) was added and incubated at 4 °C overnight, followed by washing with the PBS BSA solution. Secondary antibody (conjugated Alexa flour 488) was added and incubated at room temperature for 30 min. The slides were washed, mounted and observed using a LEICA DM6000CS microscope.

Microscopy analysis

Two-photon microscopy was performed using an LSM 7MP (Zeiss). After infection, tissues were removed and examined to identify GFP fluorescent signals. All images were acquired with the × 10 lens, and brain images were further magnified four times using the CROP function of the ZEN2012 software (Zeiss). All images were quantified using ZEN2012.

Additional information

How to cite this article: Sun, T.-S. et al. Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis. Nat. Commun. 5:5550 doi: 10.1038/ncomms6550 (2014).

References

Kim, B. E., Nevitt, T. & Thiele, D. J. Mechanisms for copper acquisition, distribution and regulation. Nat. Chem. Biol. 4, 176–185 (2008).
Article CAS Google Scholar
Samanovic, M. I., Ding, C., Thiele, D. J. & Darwin, K. H. Copper in microbial pathogenesis: meddling with the metal. Cell Host Microbe 11, 106–115 (2012).
Article CAS Google Scholar
Gutteridge, J. M., Rowley, D. A., Halliwell, B., Cooper, D. F. & Heeley, D. M. Copper and iron complexes catalytic for oxygen radical reactions in sweat from human athletes. Clin. Chim. Acta 145, 267–273 (1985).
Article CAS Google Scholar
White, C., Lee, J., Kambe, T., Fritsche, K. & Petris, M. J. A role for the ATP7A copper-transporting ATPase in macrophage bactericidal activity. J. Biol. Chem. 284, 33949–33956 (2009).
Article CAS Google Scholar
Ding, C. et al. Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence. Cell Host Microbe 13, 265–276 (2013).
Article CAS Google Scholar
Festa, R. A., Helsel, M. E., Franz, K. J. & Thiele, D. J. Exploiting Innate immune cell activation of a copper-dependent antimicrobial agent during infection. Chem. Biol. 21, 977–987 (2014).
Article CAS Google Scholar
Wolschendorf, F. et al. Copper resistance is essential for virulence of Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 108, 1621–1626 (2011).
Article CAS ADS Google Scholar
Upadhyay, S., Torres, G. & Lin, X. Laccases involved in 1,8-dihydroxynaphthalene melanin biosynthesis in Aspergillus fumigatus are regulated by developmental factors and copper homeostasis. Eukaryot Cell 12, 1641–1652 (2013).
Article CAS Google Scholar
Heitman, J. Cryptococcus: from Human Pathogen to Model Yeast ASM press (2011).
Kronstad, J. W. et al. Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box. Nat. Rev. Microbiol. 9, 193–203 (2011).
Article CAS Google Scholar
Hull, C. M. & Heitman, J. Genetics of Cryptococcus neoformans. Annu Rev. Genet. 36, 557–615 (2002).
Article CAS Google Scholar
Ding, C. et al. The copper regulon of the human fungal pathogen Cryptococcus neoformans H99. Mol. Microbiol. 81, 1560–1576 (2011).
Article CAS Google Scholar
Williamson, P. R. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J. Bacteriol. 176, 656–664 (1994).
Article CAS Google Scholar
Cox, G. M. et al. Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages. Infect. Immun. 71, 173–180 (2003).
Article CAS Google Scholar
Kronstad, J. et al. Adaptation of Cryptococcus neoformans to mammalian hosts: integrated regulation of metabolism and virulence. Eukaryot. Cell 11, 109–118 (2012).
Article CAS Google Scholar
Johnson, K. R., Zheng, Q. Y., Bykhovskaya, Y., Spirina, O. & Fischel-Ghodsian, N. A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice. Nat. Genet. 27, 191–194 (2001).
Article CAS Google Scholar
Ngamskulrungroj, P., Chang, Y., Sionov, E. & Kwon-Chung, K. J. The primary target organ of Cryptococcus gattii is different from that of Cryptococcus neoformans in a murine model. mBio 3, piie00103–12 (2012).
Article CAS Google Scholar
Waterman, S. R. et al. Role of CTR4 in the virulence of Cryptococcus neoformans. mBio 3, piie00285–12 (2012).
Article CAS Google Scholar
Schmidt, P. J., Ramos-Gomez, M. & Culotta, V. C. A gain of superoxide dismutase (SOD) activity obtained with CCS, the copper metallochaperone for SOD1. J. Biol. Chem. 274, 36952–36956 (1999).
Article CAS Google Scholar
Eisses, J. F. & Kaplan, J. H. Molecular characterization of hCTR1, the human copper uptake protein. J. Biol. Chem. 277, 29162–29171 (2002).
Article CAS Google Scholar
Nose, Y. et al. Ctr1 is an apical copper transporter in mammalian intestinal epithelial cells in vivo that is controlled at the level of protein stability. J. Biol. Chem. 285, 32385–32392 (2010).
Article CAS Google Scholar
Ohrvik, H. et al. Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain. Proc. Natl Acad. Sci. USA 110, E4279–E4288 (2013).
Article CAS Google Scholar
Ohrvik, H. & Thiele, D. J. The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy. J. Trace Elem. Med. Biol. (2014).
Agarwal, R. P. & Henkin, R. I. Zinc and copper in human cerebrospinal fluid. Biol. Trace Elem. Res. 4, 117–124 (1982).
Article CAS Google Scholar
Wagner, D. et al. Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system. J. Immunol. 174, 1491–1500 (2005).
Article CAS Google Scholar
Raja, M. R. et al. A copper hyperaccumulation phenotype correlates with pathogenesis in Cryptococcus neoformans. Metallomics 5, 363–371 (2013).
Article CAS Google Scholar
Waterman, S. R. et al. Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans. J. Clin. Invest. 117, 794–802 (2007).
Article CAS Google Scholar
Ding, C., Festa, R. A., Sun, T. S. & Wang, Z. Y. Iron and copper as virulence modulators in human fungal pathogens. Mol. Microbiol. 93, 10–23 (2014).
Article CAS Google Scholar
Lin, X., Huang, J. C., Mitchell, T. G. & Heitman, J. Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATalpha allele enhances filamentation. PLoS Genet. 2, e187 (2006).
Article Google Scholar
O'Meara, T. R. et al. The Cryptococcus neoformans Rim101 transcription factor directly regulates genes required for adaptation to the host. Mol. Cell. Biol. 34, 673–684 (2014).
Article Google Scholar
De Feo, C. J., Aller, S. G., Siluvai, G. S., Blackburn, N. J. & Unger, V. M. Three-dimensional structure of the human copper transporter hCTR1. Proc. Natl Acad. Sci. USA 106, 4237–4242 (2009).
Article CAS ADS Google Scholar
Chun, C. D. & Madhani, H. D. Ctr2 links copper homeostasis to polysaccharide capsule formation and phagocytosis inhibition in the human fungal pathogen Cryptococcus neoformans. PLoS ONE 5, e12503 (2010).
Article ADS Google Scholar
Beaudoin, J., Laliberte, J. & Labbe, S. Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport. Microbiology 152, 209–222 (2006).
Article CAS Google Scholar
Dancis, A. et al. Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell 76, 393–402 (1994).
Article CAS Google Scholar
Marvin, M. E., Williams, P. H. & Cashmore, A. M. The Candida albicans CTR1 gene encodes a functional copper transporter. Microbiology 149, 1461–1474 (2003).
Article CAS Google Scholar
Nose, Y., Kim, B. E. & Thiele, D. J. Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function. Cell Metab. 4, 235–244 (2006).
Article CAS Google Scholar
Zhou, H., Cadigan, K. M. & Thiele, D. J. A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster. J. Biol. Chem. 278, 48210–48218 (2003).
Article CAS Google Scholar
Ding, C. & Butler, G. Development of a gene knockout system in Candida parapsilosis reveals a conserved role for BCR1 in biofilm formation. Eukaryot. Cell 6, 1310–1319 (2007).
Article CAS Google Scholar
Kwon-Chung, K. J., Varma, A., Edman, J. C. & Bennett, J. E. Selection of ura5 and ura3 mutants from the two varieties of Cryptococcus neoformans on 5-fluoroorotic acid medium. J. Med. Vet. Mycol. 30, 61–69 (1992).
Article CAS Google Scholar
Staab, J. F. & Sundstrom, P. URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes. Trends Microbiol. 11, 69–73 (2003).
Article CAS Google Scholar
Lay, J. et al. Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies. Infect Immun. 66, 5301–5306 (1998).
CAS PubMed PubMed Central Google Scholar
Varma, A., Edman, J. C. & Kwon-Chung, K. J. Molecular and genetic analysis of URA5 transformants of Cryptococcus neoformans. Infect Immun. 60, 1101–1108 (1992).
CAS PubMed PubMed Central Google Scholar
Dancis, A., Haile, D., Yuan, D. S. & Klausner, R. D. The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J. Biol. Chem. 269, 25660–25667 (1994).
CAS PubMed Google Scholar
Tseng, H. K. et al. Identification of genes from the fungal pathogen Cryptococcus neoformans related to transmigration into the central nervous system. PLoS ONE 7, e45083 (2012).
Article CAS ADS Google Scholar
He, X. et al. Virulence factors identified by Cryptococcus neoformans mutant screen differentially modulate lung immune responses and brain dissemination. Am. J. Pathol. 181, 1356–1366 (2012).
Article CAS Google Scholar
Ransohoff, R. M. & Brown, M. A. Innate immunity in the central nervous system. J. Clin. Invest. 122, 1164–1171 (2012).
Article CAS Google Scholar
Zhou, Q., Gault, R. A., Kozel, T. R. & Murphy, W. J. Protection from direct cerebral cryptococcus infection by interferon-gamma-dependent activation of microglial cells. J. Immunol. 178, 5753–5761 (2007).
Article CAS Google Scholar
Brambilla, R. et al. Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury. J. Exp. Med. 202, 145–156 (2005).
Article CAS Google Scholar
Ayton, S., Lei, P. & Bush, A. I. Metallostasis in Alzheimer's disease. Free Radic. Biol. Med. 62, 76–89 (2013).
Article CAS Google Scholar
Hua, J., Meyer, J. D. & Lodge, J. K. Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans. Clin. Diagn. Lab. Immunol. 7, 125–128 (2000).
CAS PubMed PubMed Central Google Scholar
Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T. & Perfect, J. R. Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J. Bacteriol. 175, 1405–1411 (1993).
Article CAS Google Scholar
O'Meara, T. R. et al. Interaction of Cryptococcus neoformans Rim101 and protein kinase A regulates capsule. PLoS Pathog. 6, e1000776 (2010).
Article Google Scholar
Casadevall, A. et al. Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies. Antimicrob. Agents Chemother. 42, 1437–1446 (1998).
Article CAS Google Scholar
Hoebeeck, J., Speleman, F. & Vandesompele, J. Real-time quantitative PCR as an alternative to Southern blot or fluorescence in situ hybridization for detection of gene copy number changes. Methods Mol. Biol. 353, 205–226 (2007).
PubMed Google Scholar
Wang, Y. et al. Two major inositol transporters and their role in cryptococcal virulence. Eukaryot. Cell 10, 618–628 (2011).
Article CAS Google Scholar
Sabiiti, W. et al. Efficient phagocytosis and laccase activity affect the outcome of HIV-associated cryptococcosis. J. Clin. Invest. 124, 2000–2008 (2014).
Article CAS Google Scholar
Ding, C. et al. Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans. PLoS ONE 6, e28151 (2011).
Article CAS ADS Google Scholar

Download references

Acknowledgements

We thank Dr Ying-Lien Chen for providing the H99 strain. J.-Y.L. acknowledges Dr Peter Williamson (NIH, USA) for providing the ctr4Δ::URA5 and CTR4 complementation strains. C.D. acknowledges Dr Arturo Casadevall (Albert Einstein College of Medicine, USA) for providing the 18B7 antibody. C.D. acknowledges supports from the Natural Science Foundation of China (31300974) and the Fundamental Research Funds for Central Universities of China (N130120002). D.J.T. acknowledges the support from the United States National Institutes of Health grant GM041840.

Author information

Tian-Shu Sun and Xiao Ju: These authors contributed equally to this work

Authors and Affiliations

College of Life and Health Sciences, Northeastern University, ShenYang, 110015, LiaoNing, China
Tian-Shu Sun, Xiao Ju, Hui-Ling Gao, Tao Wang, Jia-Yi Li, Zhan-You Wang & Chen Ding
Department of Pharmacology and Cancer Biology, Department of Biochemistry, Duke University School of Medicine, Durham, 27710, North Carolina, USA
Dennis J. Thiele
Neural Plasticity and Repair Unit, Wallenberg Neuroscience Center, Lund University, BMC A10, 22184, Lund, Sweden
Jia-Yi Li

Authors

Tian-Shu Sun
View author publications
You can also search for this author in PubMed Google Scholar
Xiao Ju
View author publications
You can also search for this author in PubMed Google Scholar
Hui-Ling Gao
View author publications
You can also search for this author in PubMed Google Scholar
Tao Wang
View author publications
You can also search for this author in PubMed Google Scholar
Dennis J. Thiele
View author publications
You can also search for this author in PubMed Google Scholar
Jia-Yi Li
View author publications
You can also search for this author in PubMed Google Scholar
Zhan-You Wang
View author publications
You can also search for this author in PubMed Google Scholar
Chen Ding
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Z.-Y.W., D.J.T. and C.D. conceived the project. T.-S.S. and C.D. designed the study. T.-S.S., X.J., H.-L.G., J.-Y.L. and T.W. performed the experiments. T.-S.S. and C.D. participated in data analysis. Z.-Y.W., D.J.T. and C.D. wrote the manuscript.

Corresponding authors

Correspondence to Zhan-You Wang or Chen Ding.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures, Table and References

Supplementary Figures 1-6, Supplementary Table 1 and Supplementary References (PDF 16416 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sun, TS., Ju, X., Gao, HL. et al. Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis. Nat Commun 5, 5550 (2014). https://doi.org/10.1038/ncomms6550

Download citation

Received: 08 May 2014
Accepted: 13 October 2014
Published: 24 November 2014
DOI: https://doi.org/10.1038/ncomms6550

This article is cited by

Microglia are not protective against cryptococcal meningitis
- Sally H. Mohamed
- Man Shun Fu
- Rebecca A. Drummond
Nature Communications (2023)
Metal–ion promiscuity of microbial enzyme DapE at its second metal-binding site
- Atanuka Paul
- Sabyashachi Mishra
JBIC Journal of Biological Inorganic Chemistry (2021)
Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence
- Yanjian Li
- Hailong Li
- Chen Ding
Communications Biology (2019)
Copper signalling: causes and consequences
- Julianna Kardos
- László Héja
- Katalin Jemnitz
Cell Communication and Signaling (2018)
Copper import in Escherichia coli by the yersiniabactin metallophore system
- Eun-Ik Koh
- Anne E Robinson
- Jeffrey P Henderson
Nature Chemical Biology (2017)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.