Fbp1-mediated ubiquitin-proteasome pathway controls Cryptococcus neoformans virulence by regulating fungal intracellular growth in macrophages.

Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages Tong-Bao Liu and Chaoyang Xue Infect. Immun. 2014, 82(2):557. DOI: 10.1128/IAI.00994-13. Published Ahead of Print 18 November 2013.

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Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages Tong-Bao Liu,a Chaoyang Xuea,b Public Health Research Institutea and Department of Microbiology and Molecular Genetics,b Rutgers University, Newark, New Jersey, USA

C

ryptococcus neoformans is a major human fungal pathogen and the causative agent of fatal cryptococcal meningoencephalitis, which is considered an AIDS-defined condition (1–5). Despite its medical importance, the treatment for cryptococcosis is limited. With increasing concerns of drug resistance and evolution of new virulent strains (6–9), there is an urgent need to understand the molecular basis of cryptococcal infection in order to discover and develop safer and more effective antifungal drugs. Against this background of clinical need are the heroic research efforts toward understanding the mechanism of Cryptococcus development and virulence that have significantly advanced the field in the past several decades. Several virulence factors, including production of polysaccharide capsule and melanin and the ability to grow at body temperature (37°C), have been well characterized (10, 11). A number of signal pathways regulating these virulence factors and important for Cryptococcus pathogenesis have also been identified (5, 12–16). Systematic genetic analysis of genes involved in fungal virulence revealed a capsule-independent antiphagocytic mechanism, as well as a number of proteins regulating fungal virulence independent of the classical virulence factors (17, 18). These studies revealed that fungal virulence is a complex trait and that new virulence-determining mechanisms remain to be explored. Controlled protein turnover is an important regulatory mechanism of cellular function in eukaryotes. Protein ubiquitination requires a concerted action that involves an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase (19). The SCF (Skp1, Cullins, and F-box proteins) E3 ubiquitin ligase-mediated ubiquitin-proteasome system (UPS) is a major protein turnover pathway that plays an important role in the regulation of a variety of cellular functions and recently emerged as an attractive drug target for human diseases (20, 21). The SCF E3 ligases have been linked to various human diseases,

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including neurodegenerative disorders and cancer (22–24). The SCF complexes have also been reported to be important for fungal regulation of cellular functions (25). Among them, the SCF(Grr1) E3 ligase complex in Saccharomyces cerevisiae has been characterized extensively. Studies revealed that Grr1 is involved in cell cycle regulation (26, 27), nutrient sensing (28, 29), and fungal morphogenesis. Grr1 homologs in other fungi, including Candida albicans (Grr1) (30), Aspergillus nidulans (GrrA) (31), and Gibberella zeae (Fbp1) (32), have also been reported. Another F-box protein, Cdc4, has also been studied in both S. cerevisiae (33–35) and C. albicans (36–38). However, besides S. cerevisiae, substrates of the UPS in many fungi remain to be identified in most cases. There are few reports on the role of the E3 ligases in virulence in human fungal pathogens, despite recent studies linking F-box proteins to virulence in several plant-pathogenic fungi (32, 39, 40). Therefore, functional study of the SCF E3 ligases and their substrates in medically important fungi may have the potential to improve our understanding of fungal virulence and could lead to novel approaches to control fungal infections. Our previous studies identified Fbp1, an F-box protein in C.

Received 9 August 2013 Returned for modification 27 August 2013 Accepted 11 November 2013 Published ahead of print 18 November 2013 Editor: G. S. Deepe, Jr. Address correspondence to Chaoyang Xue, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00994-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.00994-13

Infection and Immunity

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Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1⌬ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1⌬ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1⌬ mutant is due to its inability to leave the lung. The fbp1⌬ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.

Liu and Xue

MATERIALS AND METHODS Strains, media, and growth conditions. C. neoformans strains used in this study are listed in Table S1 in the supplemental material. Strains were grown at 30°C on yeast extract-peptone-dextrose (YPD) agar medium and synthetic (SD) medium. Strains containing genes controlled by the CTR4 promoter were grown in YPD medium supplemented with either 25 ␮M CuSO4 and 1 mM ascorbic acid or 200 ␮M bathocuproinedisulfonic acid (BCS) (43). The pCTR4-2 plasmid was kindly provided by Tamara Doering at Washington University, St. Louis, MO, while the pCN19 plasmid was provided by Joseph Heitman at Duke University. The isc1⌬ mutant and its complemented strain were kindly provided by Maurizio Del Poeta at Stony Brook University. The macrophage-like murine cell line J774 was grown in 10-cm petri dishes in liquid Dulbecco modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal calf serum (FBS) (ATL Biologicals), 10% NCTC-109 (Gibco), and 1% nonessential amino acids (MP Biomedicals). All other media were prepared as described previously (41, 44, 45). Assay for fungal strain sensitivity to SDS and proteasome inhibitors. For SDS sensitivity testing, SDS was added to YPD to a final concentration of 0.2% (wt/vol), and 2⫻ serial dilutions were prepared in 96-well plates. Cells of H99, the fbp1⌬ mutant, and its complemented strain were added to the wells to a final optical density at 600 nm (OD600) of 0.01. The plates were kept at 30°C, and the OD600 was measured every 24 h. For inhibitor assays, MG132 (Sigma-Aldrich) or PS-341 (LC Laboratories) was added to the YPD liquid medium with 0.025% SDS to a final concentration of 100 ␮M or 200 ␮M, respectively. Twofold serial dilutions were prepared in 96-well plates. The wild-type H99 strain was added to the well containing proteasome inhibitors (MG132 or PS-134) and SDS to a final OD600 of 0.01. The fbp1⌬ mutant was added to YPD with 0.025% SDS only to a final OD600 of 0.01. The growth of cultures was determined by measuring the OD600 every 24 h. Cryptococcus-macrophage interaction assay. Macrophage-like J774 cells were cultured in DMEM with 10% heat-inactivated FBS at 37°C with 5% CO2. A total of 5 ⫻ 104 J774 cells in 0.5 ml fresh DMEM were added to each well of a 48-well culture plate and incubated at 37°C in 5% CO2 overnight. To activate macrophage cells, 50 units/ml gamma interferon (IFN-␥; Invitrogen) and 1 ␮g/ml lipopolysaccharide (LPS; Sigma) were added to each well. C. neoformans overnight cultures were washed with phosphate-buffered saline (PBS) twice and opsonized with 20% mouse complement. A total of 2 ⫻ 105 Cryptococcus cells were added to each well (yeast/J774 cell ratio, 4:1). To assess intracellular proliferation of C. neoformans, nonadherent extracellular yeast cells were removed by washing with fresh DMEM after 2 h of coincubation, and cultures were incubated for another 0, 2, or 22 h. At the indicated time points, the medium in each well was replaced by distilled water (dH2O) to lyse macrophage cells for 30

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min at room temperature. The lysate was spread on YPD plates, and the number of CFU was counted to determine intracellular proliferation. For time-lapse movie production, activated macrophage cells (2 ⫻ 105) and opsonized Cryptococcus cells (2 ⫻ 106) were coincubated in a 30-mm MatTek glass-bottom dish (coated). After 2 h of incubation, the culture was washed with fresh DMEM twice to remove detached yeast cells and replaced by 2 ml fresh DMEM. Seventeen-hour time-lapse movies with 2 min per frame were taken using a Nikon Eclipse AIRS confocal microscope. A total of 15 different views were taken for every 2 min. Resulting movies were analyzed using the software Nis Elements Viewer (Nikon). All statistical analysis was undertaken using the Student t test. P values of ⬍0.001 were considered statistically significant. qRT-PCR. Cryptococcus cells from overnight culture or mating cultures were collected. Total RNA extraction and first-strand cDNA synthesis were performed as described previously (41). Expression of ISC1 and GAPDH was analyzed using SYBR advantage QPCR premix reagents (Clontech). Gene expression levels were normalized using the endogenous control gene GAPDH, and the relative levels were determined using the comparative threshold cycle (CT) method (46). Quantitative real-time PCRs (qRT-PCRs) were performed using an Mx4000 QPCR system (Stratagene) as previously described (47). Generation of tagged protein strains. The FBP1 full-length cDNA was amplified with primers CX225 and CX443. The FBP1 cDNA lacking the F-box domain was amplified by overlap PCR using primers CX198CX199 and CX225-CX443. Both fragments were cloned into the BamHI/ NotI sites of a vector containing the Cryptococcus actin promoter (48) and a Flag epitope, generating plasmids pCXU115 and pCXU117, which contain FBP1:Flag and FBP1⌬F:Flag fusions, respectively. The above plasmids were biolistically transformed into the fbp1⌬ ura5⌬ strain to generate strains CUX138 and CUX135, which express Fbp1:Flag and Fbp1⌬F:Flag proteins, respectively. The ISC1 cDNA was amplified with primers CX551 and CX552 and cloned into the BamHI sites of pCTR4-2 vector (43) by use of an In-Fusion HD cloning kit (Clontech), generating the Isc1-hemagglutinin (HA) fusion plasmid pCXU170. To test the stability of Isc1 in the wild-type and fbp1⌬ mutant strain backgrounds, pCXU170 and pCXU108 were biolistically transformed into the wild-type strain and an fbp1⌬ mutant to generate strains CUX160 plus CUX167 and CUX118 plus CUX119, respectively. Protein preparation and mass spectrometry. To test the stability of Isc1 and Crk1 in the wild-type and fbp1⌬ mutant strain backgrounds, Isc1:HA-tagged strains CUX167 and CUX168 and Crk1:HA-tagged strains CUX118 and CUX119 were grown to mid-logarithmic phase in YPD, transferred to YPD with 25 ␮M CuSO4 and 1 mM ascorbic acid, and further incubated for the indicated amount of time. Protein extracts were prepared as described by Bahn et al. (48). Isc1:HA was detected by Western blotting using a monoclonal anti-HA antibody (GenScript). The purification of Flag-tagged Fbp1 was done using EZview Red antiFlag M2 affinity gel (Sigma-Aldrich). Cells were grown in YPD, harvested and washed with ice-cold water, and transferred into 2-ml bead-beating tubes containing 600 ␮l acid-washed glass beads. The proteins were extracted in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) by lysing cells at 4°C with glass beads (four times for 20 s each) in a FastPrep FP120 apparatus (MP Biomedical). The protein extracts were collected after 15 min of centrifugation at 14,000 rpm at 4°C. Flag-tagged proteins were affinity purified under native conditions. Protein elutions were prepared by using SDS-PAGE sample buffer without reducing agents such as 2-mercaptoethanol or dithiothreitol (DTT). To test the interaction between Fbp1 and Isc1 in vivo, Flag-tagged proteins were purified from strains CUX160, CUX140, and CUX141 by using anti-Flag affinity gel and then analyzed by immunoblotting with anti-Flag and anti-HA antibodies, respectively. To identify the proteins interacting with Fbp1, the Flag-tagged strains CUX138 and CUX135 were cultured in YPD medium, and the Flag-tagged proteins were purified using anti-Flag affinity gel and sent to the Center for Advanced Proteom-



neoformans that is essential for fungal virulence in animal models, despite the fact that the fbp1⌬ mutant produces normal virulence factors (melanin, capsule, and the ability to grow at body temperature) (41). Hence, we hypothesize that the function of Fbp1 may represent a novel virulence mechanism. In this study, we investigated the functional interaction of Fbp1 and its substrates during Cryptococcus-host interaction. Our study confirmed that Fbp1 is a part of the SCF E3 ligase complex. A detailed analysis of the fbp1⌬ mutant revealed that mutant cells remained at a low but persistent level in the infected lung. By applying proteomic techniques combined with genetic analysis, we proceeded to identify substrates of SCF(Fbp1) E3 ligase, which could be a key to understanding how the UPS regulates fungal virulence. This study showed that inositol phosphosphingolipid-phospholipase C1 (Isc1) is a substrate of Fbp1. Isc1 is required for full fungal virulence, as reported previously (42). Overall, our studies reveal a novel regulatory connection between the SCF(Fbp1) E3 ligase and inositol sphingolipid biosynthesis in C. neoformans.

E3 Ligases and Cryptococcus Virulence

infection. Female A/Jcr mice were inoculated intranasally with H99 and the fbp1⌬ mutant strain, using 105 cells per mouse. Infected lungs and brains were harvested at 3, 7, and 15 days postinoculation and at the end time point (ETP). Fungal burdens in organs infected by the fbp1⌬ mutant were compared with those in organs infected by H99. The number of yeast CFU per gram of fresh organ was measured in brain and lung homogenates. Each error bar indicates the standard error of the mean for values from three mice. (B) H&E-stained slides were prepared from cross sections of infected lungs at 3, 7, and 15 days postinfection and the end time point and visualized by light microscopy. Arrows indicate yeast cells.

ics Research at Rutgers for mass spectrometry (liquid chromatographytandem mass spectrometry [LC-MS/MS]) analysis. Virulence studies. Yeast strains were grown at 30°C overnight, and cultures were washed twice with PBS and resuspended at a final concentration of 2 ⫻ 106 CFU/ml. Groups of 10 female A/Jcr mice (NCI-Frederick) were infected intranasally with 105 yeast cells of each strain as previously described (49). For the intravenous injection model, mice were inoculated with 5 ⫻ 104 cells via tail vein injection. Over the course of the experiments, animals that appeared moribund or in pain were sacrificed by CO2 inhalation. Survival data from the murine experiments were statistically analyzed between paired groups by using the log rank test in Prism 4.0 (GraphPad Software) (P values of ⬍0.01 were considered significant). Histopathology and fungal burdens in infected organs. Infected animals were sacrificed at designated time points and the endpoint of the experiment according to the Rutgers Institutional Animal Care and Use Committee (IACUC)-approved animal protocol. Infected lungs and brains were isolated, fixed in 10% formalin solution, and sent to the Rutgers Histology Core Facility. Tissue slides were stained with hematoxylin and eosin (H&E) and examined by light microscopy. Infected lungs, brains, and spleens were also isolated and homogenized in 1⫻ PBS by use of a homogenizer. Resuspensions were diluted, 100 ␮l of each dilution was spread on YPD medium with ampicillin and chloramphenicol, and numbers of colonies were determined after 3 days of incubation. All statistical analyses were undertaken using the Student t test. P values of ⬍0.05 were considered statistically significant. Ethics statement. The animal studies conducted at Rutgers University were in full compliance with all of the guidelines set forth by the IACUC and in full compliance with the United States Animal Welfare Act (public law 98-198). The Rutgers IACUCs approved all of the vertebrate studies. The studies were conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

RESULTS

Fbp1 is required for Cryptococcus transmigration. Our previous results showed that the fbp1⌬ null mutant is hypovirulent in a murine model of systemic infection (41). Mice infected by the mutant strain are asymptomatic even after 60 days postinfection, in contrast to the average survival rate of 20 to 25 days when infected by wild-type H99. No yeast cells were recovered from the brains or spleens of mice that were infected by the fbp1⌬ mutant,


and only ⬃300 yeast CFU in each lung were recovered at 60 days postinfection, compared to ⬃108 CFU in wild-type infection at the endpoint of the infection. To better understand the dynamic of the fbp1⌬ mutant-host interaction during the infection process, fungal burdens in infected lungs were examined at 3, 7, 15, and 50 days postinoculation. Our results showed that fungal cells in fbp1⌬ mutant-infected lungs remained at a persistently low level (⬃103 CFU/g fresh lung) throughout the infection process (Fig. 1A). Fungal lesion development in the lung was also visualized in H&E-stained slides. As shown in Fig. 1B, the wild-type strain H99 caused severe damage in infected lungs, with abundant yeast cells, as early as 3 days postinoculation. In contrast, lungs infected by the fbp1⌬ mutant showed little damage, with very few yeast cells observed at different time points and predominantly localized intracellularly. Studies with animal models in vivo demonstrated that the fbp1⌬ mutant cannot disseminate to infect other organs following pulmonary infection with 105 cells. To better understand the role of Fbp1 in fungal dissemination, we applied a murine intravenous injection model of cryptococcosis to investigate whether the mutant simply cannot leave the lung or can leave the lung but cannot cross the blood-brain barrier (BBB) to cause central nervous system (CNS) infection. Our results showed that the fbp1⌬ mutant still caused lethal infection but had significant virulence attenuation compared to the wild type and the complemented strain (fbp1⌬ strain plus FBP1) (Fig. 2A). All mice (n ⫽ 5) infected by H99 or the complemented strain died at around 7 to 8 days postinjection. Three mice infected by the mutant died at 15 to 25 days postinjection, while the other two fbp1⌬ mutant-infected mice were still alive and had no sign of disease even at 37 days postinjection, when we terminated the experiment. Because both mouse groups infected by H99 or the complemented strain were terminated at around 7 days, we isolated organs from three mice infected by the mutant at 7 days postinfection for comparison. Our analysis of yeast CFU counts and lesion development in infected brains showed that the fbp1⌬ mutant could still cause infection in the brain, but the numbers of CFU were significantly reduced compared to those in mice infected by the wild-type strain (Fig. 2B

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FIG 1 The fbp1⌬ mutant remains at a persistent level in infected lungs. (A) Fungal burdens in fbp1⌬ mutant-infected lungs remain at a persistent level during

Liu and Xue

and C). At the endpoint for fbp1⌬ mutant-infected mice, three sick mice contained comparable numbers of CFU in both brains and lungs, while the two remaining, nonsymptomatic mice at 37 days postinfection contained significantly smaller fungal burdens in both brains and lungs (Fig. 2B and C). Our results indicate that Fbp1 is required but not essential for dissemination from the bloodstream to the brain. Hence, our data demonstrate that fbp1⌬ mutants could not leave the infected lung when the mice were infected via nasal inhalation.

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FIG 2 Fbp1 is required for Cryptococcus dissemination into the brain. (A) Survival curves for infected mice in a murine intravenous injection model. Female A/Jcr mice were inoculated via tail vein injection with the following strains: H99, the fbp1⌬ mutant, and its complemented strain (fbp1⌬⫹FBP1). Mice were infected with 5 ⫻ 104 yeast cells of each strain. (B and C) Brains and lungs from five mice infected with H99 or the fbp1⌬ mutant were isolated at the end time point of infection. Because all H99-infected mice died at around 7 days postinfection, three fbp1⌬ mutant-infected mice were also terminated for detection of fungal burdens in brains and lungs at 7 days postinfection (fbp1⌬ 7d) for comparison with the fungal burdens in H99-infected mice. The isolated organs were homogenized and spread on YPD plates containing ampicillin and chloramphenicol after dilution for CFU measurement.

Fbp1 is required to proliferate inside macrophages. Because the CFU results showed that the fbp1⌬ mutant remains at a persistent level in infected lungs throughout the course of intranasal infection, we hypothesized that fbp1⌬ mutants may have a defect in proliferation inside macrophages and that extracellular fungal cells may not be able to grow in the hostile host environment. To investigate how deletion of the FBP1 gene influences the interaction with host cells, we performed Cryptococcus-macrophage interaction assays by using the murine J774 macrophage-like cell line in 48-well plates. Two hours after coincubation of opsonized Cryptococcus cells and activated macrophages, nonadherent extracellular yeast cells were removed by washing with fresh medium and incubated for another 0, 2, or 22 h before macrophages were lysed by H2O. Our results showed that after 2 h of incubation, the number of yeast CFU recovered from macrophages coincubated with the fbp1⌬ mutant was comparable to that for cells coincubated with the wild type or the complemented strain, suggesting a similar level of phagocytosis, which is consistent with our previous report (41). However, after 4 h of incubation, significantly fewer CFU were recovered from the fbp1⌬ mutant-interacting macrophages (P ⬍ 0.001), i.e., only 36% of the CFU recovered from macrophages infected by the wild type. The ratio was reduced to 1:26 after 24 h of incubation (Fig. 3A). Interestingly, while a significantly larger number of CFU was recovered from macrophages infected by the wild type after 24 h, recovered mutant cells remain at a persistently low level. By testing the fungal growth rate in DMEM without macrophages, we found that both the wild-type and the mutant had similar growth in this medium (Fig. 3B). These results suggest that the fbp1⌬ mutant proliferates very slowly once it is engulfed by macrophages, which could be one reason why the fbp1⌬ mutant cannot leave the lung and disseminate to the brain in the mouse systemic infection model. To better understand the role of Fbp1 in Cryptococcus-macrophage interaction, we also measured the numbers of extracellular CFU of both the wild type and the fbp1⌬ mutant after coincubation with activated macrophages for 2, 4, and 24 h. Our results showed that similar number of extracellular CFU were recovered between the wild type and the fbp1⌬ mutant after 2 and 4 h of incubation. However, the number of CFU of the fbp1⌬ mutant was only 57% that for the wild type, on average, after 24 h of coincubation (Fig. 3C). Because the wild type and the fbp1⌬ mutant had similar growth rates in DMEM, the difference in numbers of extracellular CFU between these two strains in cultures with activated macrophages may have been caused by chemicals secreted by macrophages. To test this possibility, we performed the growth assay on these strains with a spent medium from an activated macrophage culture grown under the same conditions. Our results showed that the number of CFU recovered by the fbp1⌬ mutant was ⬃63% of that for the wild-type strain, on average, after 24 h of incubation (Fig. 3D), which supports our hypothesis that compounds secreted by activated macrophages likely can inhibit the fbp1⌬ mutant’s growth during coincubation, in addition to the intracellular growth arrest of the mutant. Because the difference in intracellular growth between the wild type and the fbp1⌬ mutant was ⬎25-fold, compared to an ⬃2-fold difference in the growth in extracellular medium, we concluded that Fbp1 plays a critical role in fungal intracellular proliferation in macrophages. This notion was confirmed by our time-lapse movies showing the replication of yeast cells inside macrophages in real time. As we expected, we observed much slower proliferation of


the mutant cells than the wild-type H99 cells inside macrophages during the 17-h movie (Fig. 3E; see Movies S1 and S2 in the supplemental material). Fbp1 is a component of the SCF E3 ligase in C. neoformans. Our previous study also found a direct interaction between Fbp1 and the Skp1 homolog in C. neoformans in a yeast two-hybrid system (41), indicating that Fbp1 could also be part of an SCF E3 ligase complex. To further investigate Fbp1 function, we performed an immunoprecipitation (IP) assay to pull down Fbp1-interacting proteins. An FBP1 overexpression construct tagged with a Flag epitope at the carboxy terminus of the Fbp1 protein (Fbp1:Flag) was generated, in which Fbp1 expression is under the control of the Cryptococcus actin promoter. Using the same strategy, the Flag tag was also fused to an Fbp1 construct lacking the F-box domain (Fbp1⌬F:Flag). The Fbp1:Flag and Fbp1⌬F:Flag constructs were expressed in an fbp1⌬ mutant background. Fbp1-associating proteins were purified by IP with an anti-Flag monoclonal antibody. As a control, a strain expressing a Flag tag fused to the G␣ protein Gpa1 (Gpa1:Flag), which is functionally unrelated to Fbp1, was also generated, and the same IP method was used to purify Gpa1: Flag-binding proteins as a control. Protein pulldown results were analyzed by LC-MS/MS analysis. Based on the LC-MS/MS analysis of the IP results, 21 proteins


were identified that were observed only in the strains expressing the Fbp1:Flag or Fbp1⌬F:Flag construct, not in the Gpa1:Flag-expressing strain, suggesting that they may specifically interact with Fbp1 (Table 1). Among them were the Skp1 homologue (CNAG_00829) and the Cdc53/Cullin homologue (CNAG_06387) (Table 1), and both of them were pulled down only by Fbp1:Flag, not by Fbp1⌬F:Flag, indicating that Fbp1 likely associates with Skp1 and Cdc53 through the F-box domain to form an SCF E3 ligase complex in C. neoformans. Proteasome inhibitor treatment replicates cell integrity defect of fbp1⌬ mutants. Our previous results showed that Fbp1 is required for cell membrane integrity and that both the fbp1⌬ mutant and strains expressing the Fbp1 allele lacking the F-box domain are hypersensitive to SDS (41). To test whether SDS sensitivity is regulated by the SCF(Fbp1) E3 ligase function, we investigated the effect of proteasome inhibitors on Cryptococcus SDS sensitivity. Fungal growth rates of the wild-type H99 strain, the fbp1⌬ mutant, and its complemented strain on YPD medium containing 0.025% SDS were determined in the absence or presence of the proteasome inhibitor MG132 or PS-341. MG132 is a cell-permeative proteasome inhibitor which can reduce the degradation of ubiquitin-conjugated proteins by the 26S complex without affecting its ATPase or isopeptidase activity (50). PS-341 is a potent and reversible proteasome inhibitor that functions to degrade intracellular polyubiquitinated proteins (50). In our as-

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FIG 3 Fbp1 is important for Cryptococcus-macrophage interactions. (A) Intracellular proliferation of C. neoformans was performed in 48-well plates, using the J774 macrophage-like cell line. PBS-washed C. neoformans strains H99, the fbp1⌬ mutant, and its complemented strain (fbp1⌬⫹FBP1) were added to the activated macrophages and incubated for 2 h at 37°C with 5% CO2. Nonadherent extracellular yeast cells were then removed by washing with fresh DMEM. Macrophage cultures were incubated for an additional 0, 2, or 22 h before they were lysed by H2O for 30 min. The lysates were spread on YPD plates, and numbers of CFU were used to determine intracellular proliferation and macrophage killing. As controls, the growth of H99 and the fbp1⌬ mutant was also measured in DMEM without macrophages (B), the extracellular space of macrophage cultures (C), and a macrophage-derived spent medium (D) under the same culture conditions. The error bars indicate standard deviations. *, P ⬍ 0.001. (E) Intracellular growth of H99 and the fbp1⌬ mutant in J774 macrophage cells. The images represent single frames from Movies S1 and S2 in the supplemental material. Arrows indicate the same macrophage cell during the movie recording.

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TABLE 1 Proteins coimmunoprecipitated with Fbp1:Flag in a pulldown assay No. of peptides identified Protein function

Gpa1:Flag

Fbp1⌬F:Flag

Fbp1:Flag

CNAG_05280 CNAG_00829 CNAG_06387 CNAG_04505 CNAG_01340 CNAG_00410 CNAG_03791 CNAG_07665 CNAG_07329 CNAG_00260 CNAG_04864 CNAG_06474 CNAG_02267 CNAG_02943 CNAG_05557 CNAG_04657 CNAG_06288 CNAG_06036 CNAG_01726 CNAG_00554 CNAG_04587 CNAG_01060

0 0 0 0 7 3 4 6 3 0 2 1 0 2 0 0 1 2 0 3 2 0

Ubiquitin-protein ligase (Fbp1) Ubiquitin-protein ligase (Skp1) Ubiquitin-protein ligase (Cdc53/Cul1) Guanine nucleotide-binding protein subunit alpha (Gpa1) ATPase with bromodomain-containing protein Conserved hypothetical protein CAMKK protein kinase Conserved hypothetical protein Conserved hypothetical protein Transformer-2-beta isoform 3 Iron regulator 1 (Cir1) Heterogeneous nuclear ribonucleoprotein (HRP1) Spindle assembly checkpoint protein SLDB Cytoplasmic protein (Slm1) Mitochondrial outer membrane protein Short-chain dehydrogenase RNA binding protein LolT-1 Transcriptional elongation regulator Inositol phosphorylsphingolipid-phospholipase C (Isc1) Conserved hypothetical protein Conserved hypothetical protein

0 0 0 19 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

26 0 0 0 4 11 13 7 3 5 4 5 4 5 2 2 1 3 1 1 2 2

26 4 32 0 23 7 1 7 9 4 4 4 4 4 3 3 3 3 5 3 1 1

says, the fbp1⌬ mutant had growth defects, while the wild type and the complemented strain exhibited normal growth on YPD agar medium containing 0.025% SDS, as expected (Fig. 4B). Neither 25 ␮M MG132 nor 100 ␮M PS-341 alone affected the growth of the wild-type strain or the mutant (Fig. 4C to E). Interestingly, when grown on YPD medium containing both SDS and a proteasome inhibitor (either MG132 or PS-341), the wild type and the complemented strain showed a significant growth defect that was similar to the growth rate of the fbp1⌬ mutant in YPD with SDS alone

(Fig. 4D). Our results thus demonstrate that treatments with proteasome inhibitors lead to the SDS hypersensitivity of wild-type Cryptococcus, which mimics that of the fbp1⌬ mutant, strongly suggesting that Fbp1 is part of the ubiquitin-proteasome system and likely regulates cell membrane integrity through its E3 ligase function. Therefore, we expect that certain substrates of Fbp1 may also participate in regulation of cell membrane integrity, which allows us to evaluate Fbp1 substrate candidates based on this phenotype. Proteins interacting with Fbp1. Because E3 ligases usually in-

FIG 4 Cryptococcus treated with proteasome inhibitors developed an fbp1⌬ mutant-like SDS sensitivity phenotype. The growth of wild-type strain H99, the

fbp1⌬ mutant, and its complemented strain (fbp1⌬⫹FBP1) in YPD liquid medium (A), YPD with 0.025% SDS (B), 25 ␮M MG132 (C), a combination of SDS and MG132 (D), 100 ␮M PS-341 (E), or a combination of SDS and PS-341 (F) is presented as OD600 measurements. In panels D and F, only the wild-type and complemented strains were treated with proteasome inhibitors. Error bars indicate standard deviations for three repeats.

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Protein ID

No. of PEST domains


wild-type H99 grown on YPD medium was arbitrarily set as 1 for comparison. (B) Cultures of mutants were grown overnight in YPD and diluted to an OD600 of 2.0. Tenfold serial dilutions were prepared, and 5 ␮l of each was plated on YPD with SDS, calcofluor white (CFW), or Congo red. Plates were incubated for 2 days at 30°C.

teract with phosphorylated substrates for ubiquitination and eventual degradation, we also analyzed other proteins that were pulled down by Fbp1 in the IP analysis to identify potential Fbp1 substrates. Besides the SCF(Fbp1) E3 ligase components, 18 other proteins interacted with both Fbp1:Flag and Fbp1⌬F:Flag in our IP pulldown and LC-MS/MS analyses (Table 1). Those proteins were selected as potential substrate candidates. The protein sequence of an E3 ligase substrate usually contains the PEST domain, a sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T), which is a signature of the short-half-life proteins that are commonly targeted for degradation by the ubiquitin-proteasome system (51). Among the 18 proteins identified, 12 have putative PEST domains in their protein sequences, based on the ePESTFind program (http://emboss .bioinformatics.nl/cgi-bin/emboss/epestfind), including the inositol phosphorylsphingolipid phospholipase C1 Isc1 and the iron regulator Cir1 that have been reported previously (42, 52, 53). Isc1 in C. neoformans is an enzyme involved in inositol sphingolipid metabolism, as it stimulates the activity to break down inositol phosphorylceramide (IPC) into phytoceramide and phosphorylinositol (42, 52). Isc1 has been found to be required for intracellular growth of Cryptococcus in macrophages (42), a function shared by Fbp1. Isc1 has three putative PEST domains, suggesting that it could be a target of the ubiquitin-proteasome pathway. Cir1 is an iron-responsive transcription factor that controls the regulation of genes for iron acquisition and the known major virulence factors of the pathogen, including capsule and melanin production and the ability to grow at body temperature (37°C) (53). A previous report indicated that Cir1 is degraded by a ubiquitin-proteasome system, but the nature of the potential E3 ligase remains unknown (54). Cir1 contains two putative PEST domains. Hence, our results suggest that Isc1 and Cir1 may be substrates of the SCF(Fbp1) E3 ligase. However, the involvement of Cir1 in the development of major virulence factors is different from the Fbp1 function (53). Therefore, the posttranslational regulation of Cir1 may be more complex. We focus on the Isc1 protein in this study. Proper expression of Isc1 is required for normal fungal growth on SDS medium. Because a substrate of Fbp1 will likely be accumulated in an fbp1⌬ mutant background due to a lack of proper ubiquitination and degradation, we generated ISC1 overexpression strains in which ISC1 expression is under the control of the Cryptococcus actin promoter. Compared to its expression in


the wild type, the expression of ISC1 in the overexpression strains was increased over 300-fold as detected by qRT-PCR (Fig. 5A). To fully evaluate the function of Isc1, we also utilized the isc1⌬ mutant for our study. We then examined the potential role of Isc1 in regulating cell membrane integrity by examining the SDS sensitivity phenotype of the ISC1 overexpression strains and the isc1⌬ mutant cells. We indeed observed that all overexpression strains were sensitive to SDS treatment, as they had growth defects in medium containing 0.025% SDS. We also found that these strains grew normally in the presence of the cell wall-destabilizing agents calcofluor white (CFW) and Congo red, which is consistent with phenotypes of the fbp1⌬ mutant (Fig. 5B). We found that the isc1⌬ mutant was sensitive to SDS as well (Fig. 5B). The SDS sensitivity phenotype of both null mutants and overexpression strains of Isc1 suggests that the expression of Isc1 is tightly regulated and that proper expression is necessary for its normal cellular function. The phenomenon that deletion or overexpression of a target gene leads to similar phenotypes has also been documented in other organisms, as described in a recent review (55). The stability of Isc1 is dependent on Fbp1 function. To further examine the potential interaction between Isc1 and Fbp1, we generated an Isc1 expression construct in the vector pCTR4-2 in which Isc1 was fused with an HA tag at its C terminus and was under the control of an inducible CTR4 promoter that is induced by BCS and repressed by copper (43). The construct expressing the Isc1:HA fusion protein was introduced into an Fbp1:Flag overexpression strain and the fbp1⌬ mutant background. The total protein from the strain expressing both Fbp1:Flag and Isc1:HA was purified using EZview Red anti-Flag M2 affinity gel (Sigma, St. Louis, MO) and immunoblotted with anti-Flag and anti-HA antibodies. Both Flag and HA signals were detected from the co-IP product, demonstrating that Isc1 indeed interacts with Fbp1 in C. neoformans (Fig. 6A). To evaluate our hypothesis that Isc1 is an Fbp1 substrate, we examined whether the stability of the Isc1 protein is dependent on SCF(Fbp1) E3 ligase function. The Isc1:HA fusion construct was expressed in the wild-type H99 strain and the fbp1⌬ mutant, and the stability of the Isc1:HA protein was examined in these strain backgrounds. Strains expressing the Isc1:HA fusion protein were first grown in YPD medium with 200 ␮M BCS to induce the CTR4 promoter and then washed with PBS. Washed cultures were transferred to YPD containing 25 ␮M CuSO4 and 1 mM ascorbic acid to block the transcription of ISC1:HA. Cells were collected after 0,

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FIG 5 Isc1 is required for cell integrity. (A) Relative qRT-PCR analysis was performed to measure the overexpression of ISC1. The gene expression level in

Liu and Xue

Fbp1:Flag and Isc1:HA were extracted. The potential Fbp1-Isc1 interaction was analyzed by co-IP with anti-Flag or anti-HA antibody and evaluated by immunoblotting. (B) The stability of the Isc1 protein is dependent on the expression of Fbp1. The PCTR4-ISC1:HA construct was expressed in either the wild-type (WT) or fbp1⌬ mutant background. Cells were grown in YPD to the mid-logarithmic phase. Cells were harvested after CuSO4 addition to stop ISC1 transcription at the indicated times, and the abundance of Isc1 was monitored by immunoblotting using HA antibody. The expression of the actin gene was used as a loading control. (C) Isc1 accumulates in the fbp1⌬ mutant background. The PISC1-ISC1:HA construct was expressed under the control of its native promoter in either the wild-type or fbp1⌬ mutant background. Cells were harvested after being cultured in YPD overnight, and the abundance of Isc1 was monitored by immunoblotting (WB) as described above. (D) Southern blot analysis to determine the copy number and integrity of the PISC1-ISC1:HA plasmid in the genomic DNAs of H99 and the fbp1⌬ mutant strain.

1, 2, and 4 h of incubation, and the abundance of the Isc1:HA protein was measured by Western blotting. In our assays, the Isc1:HA protein was degraded in a time-dependent manner over the period examined (0 to 4 h) in the wild-type background, but it was relatively stable in the fbp1⌬ mutant, indicating that the stability of Isc1:HA is dependent on Fbp1 (Fig. 6B). To test whether Isc1 accumulated in the fbp1⌬ mutant background, another Isc1:HA fusion construct in which ISC1 was controlled by its native promoter was made and transformed into H99 and the fbp1⌬ mutant background. The abundance of the Isc1:HA protein was measured by Western blotting. The signal of Isc1:HA in the fbp1⌬ mutant background was much stronger than that in the wild-type strain, but it was weaker than that in strains expressing Isc1:HA under the control of the CTR4 promoter (Fig. 6C). The presence of a single ectopic copy of the ISC1:HA gene in these strains was confirmed by Southern blotting (Fig. 6D). This result demonstrates that Isc1 is stabilized in the fbp1⌬ mutant background. Therefore, we conclude that Isc1 is a substrate of Fbp1 in C. neoformans. Isc1 is required for fungal virulence. Isc1 has been reported to play a key role in protecting C. neoformans from the intracellular environment of macrophages and is important for fungal dissemination to the central nervous system and the development of meningoencephalitis (42). Since Fbp1 is also required for Cryptococcus-macrophage interaction and for fungal dissemination, we further investigated the connection between the Fbp1 E3 ligase and Isc1. As an Fbp1 substrate, the Isc1 protein is stabilized in an

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fbp1⌬ mutant background, based on our study. Therefore, we investigated the role of Isc1 in fungal virulence by using both the isc1⌬ mutant and its overexpression strain. In a murine inhalation model of cryptococcosis, we observed virulence attenuation in both the isc1⌬ mutant and its overexpression strain. In accord with previous results (41), all mice infected with 105 yeast cells of wild-type strain H99 had a median survival time of 20 days due to lethal infection. In contrast, both the isc1⌬ mutant and the ISC1 overexpression strain showed significant virulence attenuation, with median survival times of 29 and 25 days, respectively (Fig. 7A). The virulence attenuation of the isc1⌬ mutant we observed is consistent with a previous report (42). To test whether an isc1⌬ mutation can partially rescue the hypovirulence of the fbp1⌬ mutant, we generated an isc1⌬ fbp1⌬ double mutant. As expected, mice infected by the isc1⌬ fbp1⌬ double mutant developed lethal infections by 40 days postinoculation (Fig. 7A), indicating that the isc1⌬ mutation indeed partially rescued the virulence attenuation of the fbp1⌬ mutant. To better understand the virulence attenuation of the Isc1related strains, we investigated the disease progression by examining the fungal burdens of infected lungs and brains in a time course study. Mouse lungs and brains infected by the wild type, the ISC1 overexpression strain, or the isc1⌬ mutant were isolated at 3, 7, and 14 days postinfection. Our results showed that lungs infected by either the isc1⌬ mutant or the ISC1 overexpression strain showed around 10 times fewer CFU at 7 and 14 days postinfection than the H99-infected lungs, but the difference was not



FIG 6 Isc1 interacts with Fbp1 and is a substrate of Fbp1. (A) Isc1 interacts with Fbp1. Proteins from cells expressing Fbp1:Flag (CUX134), Isc1:HA, or both


lular proliferation of C. neoformans was measured in 48-well plates by using the J774 macrophage-like cell line. Cryptococcal strain H99, the isc1⌬ mutant, the complemented isc1⌬ strain (isc1⌬⫹ISC1), the isc1⌬ fbp1⌬ double mutant, and the ISC1 overexpression strain (PACT-ISC1) were added to the activated macrophages and incubated for 2 h at 37°C with 5% CO2. Nonadherent extracellular yeast cells were then removed by washing with fresh DMEM. Macrophage cultures were incubated for an additional 0 or 22 h before they were lysed by H2O for 30 min. The lysates were spread on YPD plates, and numbers of CFU were used to determine intracellular proliferation and macrophage killing. The error bars indicate standard deviations for three repeats. *, P ⬍ 0.01.

FIG 7 Both Fbp1 and Isc1 are required for efficient dissemination of C. neoformans to the brain. (A) Survival curves for infected mice after intranasal infection with wild-type H99, the isc1⌬ mutant, the ISC1 overexpression strain (PACT-ISC1), the fbp1⌬ mutant, or the isc1⌬ fbp1⌬ double mutant. The survival rate was plotted against the number of days after inoculation. (B and C) Fungal burdens in mice intranasally infected by wild-type H99, the isc1⌬ mutant, or the ISC1 overexpression strain were analyzed at 3, 7, and 14 days postinfection and the end time point (ETP) of the infection. Numbers of CFU per gram of fresh organ were measured in lung (B) and brain (C) homogenates. Data points and error bars indicate the means and standard errors of the means for values from three animals. *, P ⬍ 0.01.

significant at 3 days postinfection (Fig. 7B). On the other hand, brains infected by the isc1⌬ mutant showed significantly reduced fungal burdens throughout the infection process, consistent with the previous report that Isc1 is required for dissemination (42). The fungal burdens in brains infected by the ISC1 overexpression


strain were also reduced, but to a lesser extent, consistent with the survival curve for infected mice (Fig. 7C). These outcomes again suggest that the Isc1 protein level may be tightly regulated by the Fbp1 E3 ligase, and levels that are too high or too low could lead to virulence attenuation. Isc1 is required for Cryptococcus-macrophage interaction. To better understand how Fbp1 may regulate Isc1 function in vivo, we also tested the Cryptococcus-macrophage interaction in the isc1⌬ mutant and its overexpressed strain. Our results found that both strains showed reduced intracellular growth, a phenotype observed in the fbp1⌬ mutant as well, with the ISC1 overexpression strain showing a lesser defect than the null mutant (Fig. 8). The isc1⌬ fbp1⌬ double mutant also showed a significant intracellular growth defect, but it was much less severe than that of the fbp1⌬ single mutant, which is consistent with the conclusion that Isc1 is a downstream target of Fbp1. Fbp1 and Isc1 are both required for Cryptococcus resistance against nitrosative stress. It has been reported that Isc1 is essential for Cryptococcus resistance against host stress responses, notably nitrosative stress and oxidative stress (42). To understand whether Fbp1 has similar functional properties, we performed a growth assay of the wild type and the fbp1⌬ mutant under either nitrosative or oxidative stress conditions, using a previously described method (42). Compared to the wild-type strain, the isc1⌬ mutant, fbp1⌬ mutant, isc1⌬ fbp1⌬ double mutant, and ISC1 overexpression strains all had growth defects under nitrosative stress conditions, although the sensitivities of the fbp1⌬ mutant and the ISC1 overexpression strain were not as high as that of the isc1⌬ mutant (Fig. 9). Meanwhile, oxidative stress response assays showed a less conclusive outcome. The isc1⌬ mutant and isc1⌬ fbp1⌬ double mutant showed high sensitivity to 5 mM H2O2 at low pH (pH 4.0), but the fbp1⌬ and ISC1 overexpression strains did not show significant differences compared to wild-type strain H99 (see Fig. S3 in the supplemental material). We hypothesize that the function of Isc1 in the nitrosative stress response is regulated by the Fbp1 E3 ligase, but its function in the oxidative stress response may be regulated by some other upstream factors.

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FIG 8 Isc1 is important for Cryptococcus-macrophage interactions. Intracel-

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FIG 9 Both Fbp1 and Isc1 are required for fungal resistance against nitrosative stress. Serial dilutions of the indicated strains were grown on YNB medium at pH 4.0 and supplemented or not with 1 mM NaNO2 for 48 h and 96 h before photography.

DISCUSSION

The E3 ubiquitin ligases are key enzymes in the UPS-mediated ubiquitination and degradation process. They function as the substrate recognition modules of the system and determine the specificity of protein degradation. Our previous study revealed that the F-box protein Fbp1 controls Cryptococcus virulence without affecting classical virulence factors. Here we further confirm that Fbp1 is part of an SCF E3 ligase complex that participates in regulating protein turnover. We hypothesize that the SCF(Fbp1) E3 ligase may regulate fungal pathogenesis through a mechanism that is independent of the classical virulence factors. Studies by other groups have also identified multiple proteins that regulate fungal disease development without affecting known virulence factors (17), an indication of the complexity of mechanisms that regulate fungal virulence. Hence, it is important to investigate such additional regulatory mechanisms to better understand fungal infections, which may lead to the development of new drug targets. To characterize the role of the SCF(Fbp1) E3 ligase in fungal infection, we monitored the disease progression in lungs infected by the fbp1⌬ mutant by examining the fungal burden. It is interesting that lungs infected by the mutant maintained a low but persistent level of yeast cells. Our data from Cryptococcus-macrophage interaction assays indicate that Fbp1 is required for fungal proliferation in macrophages, after phagocytosis. Hence, it is possible that the fbp1⌬ mutant cannot survive extracellularly in the lung due to its defect in cell integrity, as shown in Fig. 5 and our previous report (41), while a small population of mutant cells survive and remain in alveolar macrophage cells without active replication, leading to a persistent level of yeast cells in the lung throughout the infection process. Furthermore, the intracellular localization may prevent the mutant cells from leaving the lung to disseminate to other organs. It is also possible that the Fbp1 E3 ligase may target some host factors for degradation during infection to modify the host immune system. These surviving yeast cells could have some unique cellular properties. It will be interesting to characterize these remaining fungal cells in the lung, which may lead to a better understanding of intracellular survival mechanisms of Cryptococcus. The outcome of the macrophage killing experiment we presented in this study is somewhat different from our previous re-

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Overall, our study revealed that Fbp1 plays a key role in the survival and proliferation of cryptococcal cells inside macrophages during pulmonary infection. We identified and characterized an important substrate that is required for fungal virulence, a phenotype regulated by the SCF(Fbp1) E3 ligase-mediated ubiquitin-proteasome pathway.

port, in which we concluded that there is no significant difference between the wild type and the fbp1⌬ mutant in fungal survival in macrophages (41). These differences may due to the differences in experimental procedures. In the current study, we removed the extracellular yeast cells by washing macrophages with fresh medium after coincubating yeast cells with macrophages for 2 h. In our previous report, on the other hand, the macrophages in the wells were not washed after coincubation. Instead, both intracellular and extracellular yeast cells were collected and the number of yeast CFU determined to evaluate overall antifungal killing activity of the macrophages. Because the difference in extracellular growth between the wild type and the fbp1⌬ mutant was relatively small and the proliferation of extracellular yeast cells could affect the overall CFU counts, the conclusion based on total CFU (intracellular and extracellular) could not reflect the difference in intracellular proliferation between these two strains. Our current study clearly demonstrated that Fbp1 is required for intracellular yeast proliferation by removing the extracellular yeast cells. Hence, we consider that the results presented in this study better represent the intracellular proliferation and survival of the mutant cells in the macrophage culture. In addition, the small but clear difference in numbers of extracellular fungal CFU between the wild type and the mutant after coincubation with spent medium or activated macrophages could indicate that macrophages secrete some active compounds to inhibit the mutant’s growth. It would be interesting to identify such active compounds from the spent medium in the future. The SCF E3 ligase complexes bind to substrates that contain specific recognition signals for ubiquitin-protein ligation and degradation, thereby determining the specificity of the regulatory system. Hence, identification of its substrates is the key to understanding how an SCF(Fbp1) E3 ligase controls fungal virulence. By employing proteomic approaches and phenotypic analysis, we identified a list of candidates. Interestingly, a few of them have been studied in C. neoformans, including Isc1 (42) and Cir1 (53). It is interesting that both proteins are required for cell membrane integrity, a function shared by Fbp1. The result that the wild-type strain H99 becomes sensitive to SDS after treatment with proteasome inhibitors strongly indicates that Fbp1 regulates cell membrane integrity via its E3 ligase function. Hence, the regulation of cell integrity could be a common function of Fbp1 substrates. We identified the iron master regulator Cir1 in the Fbp1 pulldown results. It is possible that Fbp1 may regulate Cir1 function. However, Cir1 regulates fungal virulence by controlling the classical virulence factors, including production of melanin and capsule and cell growth at 37°C, which is different from the potential virulence factor-independent virulence regulation by Fbp1. We do not have evidence right now to determine whether Cir1 is a substrate of Fbp1, other than the fact that it was pulled down by Fbp1 in our IP experiment. It is possible that the SCF(Fbp1) E3 ligase is one of the regulatory mechanisms of Cir1 function. Other E3 ligases may also play a role in the ubiquitination and degradation of Cir1 protein. Therefore, the potential connection between Fbp1 and Cir1 remains to be determined. Inositol sphingolipids are required for Cryptococcus infection by regulating signal events that control the production of virulence factors (56, 57) and the regulation of phagocytosis (58, 59). Two enzymes in the inositol sphingolipid biosynthetic pathway, namely, inositol phosphorylceramide synthase (Ipc1) and inositol phosphosphingolipid-phospholipase C (Isc1), are required for


ACKNOWLEDGMENTS We thank Issar Smith, Aaron Mitchell, and Alex Idnurm for critical readings of the manuscript and valuable comments on the study. We thank Tamara Doering, Maurizio Del Poeta, and Joe Heitman for plasmids and strains. We also thank Yina Wang for assistance with animal studies. This study was supported by American Heart Association grant 12SDG9110034 to C.X.

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the intracellular growth of Cryptococcus inside macrophages (42, 60). Ipc1 utilizes phytoceramide to generate diacylglycerol (DAG) in the synthesis of inositol phosphorylceramide (IPC), which is subsequently transformed into mannosyl-inositol phosphorylceramide (MIPC) and mannosyl– di-inositol phosphorylceramide [M(IP)2C]. Isc1 generates phytoceramide in the hydrolysis of IPC, MIPC, or M(IP)2C (52). However, it remains unknown how inositol sphingolipid biosynthesis is regulated in C. neoformans. Our results demonstrate that Isc1 is a substrate of Fbp1, which uncovers a potential regulatory mechanism of sphingolipid production and breakdown. Our results show that both the isc1⌬ mutant and the ISC1 overexpression strain significantly reduced fungal virulence, although the virulence attenuation of either strain was not as severe as that of the fbp1⌬ mutant. Because the Isc1 protein should be stabilized in an fbp1⌬ mutant background, the reduced virulence in the ISC1 overexpression strain was expected. The Isc1 protein level may be tightly regulated by the SCF(Fbp1) E3 ligase, and either too high or too low a level of this protein could lead to a defective function and cause virulence attenuation. There are other reports which have shown that null mutants and overexpression strains of certain genes share similar phenotypes (55, 61, 62). The greater virulence attenuation of the fbp1⌬ mutant than the ISC1 overexpression strain suggests that Fbp1 likely has additional substrates involved in the regulation of fungal virulence. Meanwhile, both Fbp1 and Isc1 are required for fungal resistance to nitrosative stress, but only Isc1 is required for oxidative stress. Therefore, it is also possible that Isc1 may be regulated by other regulatory systems besides the SCF(Fbp1) E3 ligase. It would be interesting to investigate the functions of additional Fbp1 substrates. Besides studies of S. cerevisiae, most studies on the UPS focus on F-box proteins in fungi, and there are only a few E3 ligases with known substrates, including Sol1 (36) and Pcl5 (63) in C. albicans. Our study identified a number of potential substrates of an SCF E3 ligase in Cryptococcus, which may open up a new research venue for understanding how the UPS regulates fungal pathogenesis.

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27.

28. 29.

31.

32.

33.

34.

35.

36. 37. 38.

39.

40.

41.

42.

43. 44.

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45. 46. 47.

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49. 50. 51. 52.

53. 54.

55. 56.

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Phagocytosis of Cryptococcus neoformans by alveolar macrophages.

Catecholamines and virulence of Cryptococcus neoformans.

The tools for virulence of Cryptococcus neoformans.

Role of ferric reductases in iron acquisition and virulence in the fungal pathogen Cryptococcus neoformans.

Population genomics and the evolution of virulence in the fungal pathogen Cryptococcus neoformans.

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

Virulence-Associated Enzymes of Cryptococcus neoformans.

Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated macrophages.

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence.

Cryptococcus neoformans Thermotolerance to Avian Body Temperature Is Sufficient For Extracellular Growth But Not Intracellular Survival In Macrophages.

Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence.

Disarming Fungal Pathogens: Bacillus safensis Inhibits Virulence Factor Production and Biofilm Formation by Cryptococcus neoformans and Candida albicans.

Quorum sensing-mediated, cell density-dependent regulation of growth and virulence in Cryptococcus neoformans.

Capsule size of Cryptococcus neoformans: control and relationship to virulence.

Opsonization of Cryptococcus neoformans by human immunoglobulin G: role of immunoglobulin G in phagocytosis by macrophages.

Targets of the Sex Inducer homeodomain proteins are required for fungal development and virulence in Cryptococcus neoformans.

Growth of Cryptococcus neoformans in a thiamine-free medium.

Regulation of melanin production by Cryptococcus neoformans.

Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin.

Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans.

Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3.

Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans.

Cryptococcus neoformans var gattii.