Role of the Anaphase-Promoting Complex Activator Cdh1 in the Virulence of Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a globally distributed human fungal pathogen that can cause cryptococcal meningitis with high morbidity and mortality. In this study, we identified an anaphase-promoting complex (APC) activator, Cdh1, and examined its impact on the virulence of C. neoformans. Our subcellular localization analysis revealed that Cdh1 is situated in the nucleus of C. neoformans. Disrupting or overexpressing the CDH1 gene caused abnormal capsule formation in C. neoformans. The cdh1Δ mutant displayed slight sensitivity when grown at 37 °C, indicating that Cdh1 plays a role in maintaining the growth of C. neoformans at 37 °C. A fungal virulence assay showed that Cdh1 is closely associated with the virulence of C. neoformans, and both the cdh1Δ mutant and CDH1^OE overexpression strains significantly diminished the virulence of C. neoformans. The Cryptococcus–macrophage interaction assay revealed that both the cdh1∆ mutant and the CDH1^OE strains had significantly lower proliferation ability inside macrophages. Furthermore, the infection of the cdh1Δ mutant significantly activated neutrophil recruitment, as well as Th2 and Th17 immune responses, in lung tissue. In summary, our findings indicate that Cdh1 is crucial for producing virulence factors and fungal virulence in C. neoformans. The findings of this study can offer valuable insights and form the basis for further study of the regulatory mechanisms governing the pathogenicity of C. neoformans, potentially leading to the development of novel therapeutic strategies.

1. Introduction

Cryptococcus neoformans is an opportunistic fungal pathogen found widely in the natural environment. The desiccated spores or yeast cells from the environment can invade the human body through the respiratory tract, causing cryptococcal pneumonia. This initial infection can further disseminate via the bloodstream, resulting in systemic infections involving various organs such as skin, eyes, and bones [1,2,3,4]. Moreover, it can infiltrate the central nervous system, causing a fatal infection, cryptococcal meningitis [5]. It is estimated that C. neoformans causes about 194,000 infections and 147,000 deaths each year [6]. In 2022, the World Health Organization identified C. neoformans as a critical-priority fungal pathogen, posing a significant threat to public health worldwide [7].

As a pathogenic fungus, C. neoformans possesses a variety of classical virulence factors, such as the ability to survive at 37 °C, the production of polysaccharide capsules, and the formation of melanin. The primary prerequisite for cryptococcal infection of mammalian hosts is the ability to grow at 37 °C. The presence of polysaccharide capsules can inhibit the phagocytosis of C. neoformans by host immune cells, and the phagocytosis efficiency of immune cells decreases with an increase in capsule thickness [8,9]. Conversely, the mutant strains with an impaired capsule structure are often more susceptible to phagocytosis by macrophages [10,11,12]. The shed capsule antigen can also cause damage to the host immune system through various mechanisms [13]. Melanin can protect pathogenic fungi from phagocytosis by pulmonary macrophages by eliminating antimicrobial oxides produced in phagocytic cells, reducing host-killing and immune response ability [14]. Melanin can enhance the nonlytic exocytosis of C. neoformans, allowing it to evade phagocytic cells and decreasing the effective concentration of drugs by binding them in the cell wall, ultimately facilitating the dissemination of C. neoformans [15,16]. In addition, C. neoformans can also produce virulence factors such as urease [17], phospholipase [18,19,20], and protease [21,22]. The production of virulence factors helps C. neoformans adapt to the host environment and resist host defense responses against pathogens.

Cyclins are a class of proteins that play a crucial role in regulating the cell cycle, a process essential for maintaining genomic integrity in organisms. The dysregulation of cyclin activity has been implicated in the development of various types of cancer [23], such as breast cancer [24], myeloma [25], colon cancer [26], ovarian cancer [27], and oral cancer [28]. Additionally, cell cycle regulatory proteins have been found to contribute to the pathogenicity of human fungal pathogens [29]. For example, in C. neoformans, the G1 cyclin gene CLN1 has been identified, and its mRNA has been shown to be periodically expressed during the cell cycle [30]. Mutant strains lacking the G1/S cyclin CLN1 exhibit enlarged capsules, growth defects at higher temperatures, and an inability to synthesize melanin and capsules, ultimately resulting in a loss of pathogenicity [31,32]. Similarly, the deletion of the cyclin Cbc1 in C. neoformans significantly reduces the strain’s ability to produce virulence factors, leading to a complete loss of pathogenicity [33]. These findings underscore the critical role of cell cycle-related proteins in the evolution of virulence in fungal pathogens.

C. neoformans is an important fungal pathogen that poses a substantial threat to human and public health. The complexity inherent in the structure of fungal cell walls presents significant challenges in the treatment of fungal infections, rendering them more difficult to manage than bacterial and viral infections. Moreover, the available antifungal drugs are limited and expensive and face limitations in efficacy due to drug resistance, toxicity, and high treatment costs. This results in significant challenges in managing fungal infections [34,35], leading to high morbidity and mortality rates associated with conditions such as cryptococcosis. Understanding the pathogenic mechanism of C. neoformans has significant implications for developing new drugs and effectively treating cryptococcosis. In this study, we identified an anaphase-promoting complex activator, Cdh1, and investigated its role in the virulence of C. neoformans. Our findings indicate that Cdh1 significantly influences the formation of virulence factors and plays a crucial role in the virulence of C. neoformans.

2. Materials and Methods

2.1. Strains and Media

The strains used in this study are detailed in Table S1 [36,37]. E. coli DH5α was grown in a lysogeny broth (LB, 0.5% yeast extract, 1% tryptone, and 1% NaCl). C. neoformans strains were cultured on a YPD medium (1% yeast extract, 2% peptone, and 2% dextrose). Melanin formation was induced using a Niger seed medium, and capsule formation in C. neoformans was induced by culturing in a Sabouraud medium diluted with 50 mM MOPS at pH 7.3 [38]. All other media used in this study were prepared as previously described [39]. The strains were stored as 20% glycerol stocks at −80 °C.

2.2. Construction of Recombinant Strains

The plasmids and primers used in this study are shown in Table S2 [40,41,42,43] and Table S3. Primer synthesis and sequence determination were carried out by Sangon Biotech (Sangon Biotech, Shanghai, China). Plasmid construction was performed by homologous recombination according to the instructions of the Hieff Clone^® Plus One Step Cloning Kit (Yeasen, Shanghai, China). All the restriction enzymes used in this study were obtained from New England Biolabs (NEB, Beijing, China). PCR amplification was performed using the Hieff Canace^® Gold High Fidelity DNA Polymerase (Yeasen, Shanghai, China). C. neoformans CDH1-related strains were obtained through biolistic transformation, as described previously [44].

The construction of the cdh1Δ mutant strain involved the targeted replacement of the CDH1 gene with the NEO resistance gene through homologous recombination. Initially, the upstream (841 bp) and downstream (820 bp) flanking fragments of CDH1 were amplified from the wild-type strain H99 genome using the TL1828/TL1829 and TL1830/TL1831 primers. Simultaneously, the NEO marker gene (2098 bp) was amplified using TL17/TL18 as the primers and pJAF1 as the template. Subsequently, overlap PCR was conducted to fuse the CDH1 upstream with NEO (2194 bp) using the primer TL1828/TL20. The downstream fragment of CDH1 was fused to NEO (1823 bp) using the primer TL19/TL1831. The resulting fusion fragments were purified and concentrated to 2 μg/μL, followed by transformation into α or a mating type of wild-type C. neoformans cells using biolistic transformation. The cdh1Δ mutants were selected using 200 mg/L G418 (Sangon Biotech, Shanghai, China) and further confirmed via Southern blot analysis using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche, Mannheim, Germany). The successfully constructed α and a mating type cdh1Δ mutant strains were respectively designated as TBL508 and TBL509.

To obtain a complemented strain of the cdh1Δ mutant, a 4.1 kb fragment of the wild-type strain H99 genome was amplified with primers TL1947/TL1948 and was subsequently cloned into the BamHI site of pTBL1 to generate the recombinant plasmid pTBL404. This fragment contained a segment of approximately 0.8 kb upstream and a terminator segment of approximately 0.9 kb downstream of the coding region of the CDH1 gene. The linearized pTBL404, following digestion with XhoI, was purified and concentrated to 2 μg/μL. The linearization plasmid pTBL404 was transformed into α or a mating type of cdh1Δ mutants by biolistic transformation. After screening for 100 mg/L NAT (Qingpu, Shanghai, China) resistance and confirmation via PCR using the primers TL1945/TL1946, the obtained cdh1::CDH1 complemented strains of α and a mating types were designated as TBL512 and TBL513, respectively.

To generate a CDH1 overexpression strain (hereafter called CDH1^OE), the CDH1 gene (2370 bp) was amplified from the wild-type H99 genome using the primers TL1945/TL2192 and subsequently cloned into the BamHI site of pTBL405, yielding the CDH1: MYC fusion plasmid pTBL443 in which the expression of the CDH1 gene was under the control of an action promoter. Following linearization with ScaI, the purified pTBL443 was concentrated to 2 μg/μL and introduced into cdh1Δ mutant strains via biolistic transformation. Transformants were screened for resistance to 150 mg/L hygromycin (Sangon Biotech, Shanghai, China), followed by total protein extraction and immunoblotting using a Myc-tag Polyclonal antibody (Proteintech Group, Wuhan, China) to detect the expression of Cdh1:Myc.

To evaluate the expression level of Cryptococcus genes, we conducted qRT-PCR to verify the transcription levels of the mRNA associated with C. neoformans. Overnight cultures of each C. neoformans strain were collected and washed twice with sterile ddH₂O to obliterate the medium. Subsequently, the yeast cells were freeze-dried, and total RNA was extracted using the Omega Bio-Tek Total RNA Kit I (Norcross, GA, USA) following the manufacturer’s instructions. RNA quantification was performed using a MicroSpectrophotometer (K5600C; KAIAO, Shanghai, China), and cDNA was synthesized using the Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, Shanghai, China) with the primers TL2135/TL2136 (CDH1), TL2466/TL2467 (APP1), and TL217/TL218 (GAPDH) on the Light Cycler 96 QPCR system (Roche, Mannheim, Germany). Gene expression levels were normalized using the endogenous control gene GAPDH, and relative expression was determined using the 2^−ΔΔCT method [45]. Statistically significant differences were evaluated using Dunnett’s multiple comparison test (GraphPad Software Inc., San Diego, CA, USA). The CDH1^OE overexpression strains of the α and a mating type were named as TBL580 and TBL575, respectively.

To determine the subcellular localization of Cdh1 protein in C. neoformans, the coding region of the CDH1 gene (2373 bp) was amplified by TL2119/TL2120 primers from the wild-type strain H99 genome. The amplified gene was cloned into pCN19 to construct the GFP-Cdh1 fusion protein expression vector, designated as pTBL435. Subsequently, the constructed plasmid was linearized with SacI, concentrated to 2 µg/µL, and transformed into the cdh1Δ mutant strain via biolistic transformation. Transformants were initially screened on a YPD medium supplemented with nourseothricin sulfate (100 mg/L), and the fluorescence of the strain was further confirmed using a fluorescence microscope using a 100× objective lens (Axo observer 3; Zeiss, Oberkochen, Germany). The resulting Cdh1 green fluorescent strain was named TBL630.

2.3. Subcellular Localization Analysis of Cdh1 (DAPI Staining)

To determine the localization of Cdh1 in C. neoformans, we performed DAPI staining. Briefly, 500 μL of overnight cultures were collected and washed twice with 1 mL sterile 1 × PBS buffer (pH 7.4). Subsequently, 200 μL of 9.3% formaldehyde was added to fix the cells at room temperature for 10 min. Following fixation, the cells were washed twice with 1 mL 1 × PBS buffer. Then, the cells were resuspended in 300 μL 1 × PBS buffer, an equal volume of 1 × PBS buffer containing 1% Triton X-100 was added, and the permeabilization was allowed to proceed for 10 min at room temperature. Afterward, the reaction solution was washed twice with 1 mL of 1 × PBS buffer. Next, the cells were resuspended in 300 μL of PBS, after which we added an equal volume of 10 μg/mL DAPI (Sigma, Saint Louis, MO, USA) staining solution. After thorough mixing, the cells were incubated at 60 rpm for 15 min at room temperature. Finally, the cells were washed twice with 1 mL of PBST buffer, resuspended, and observed with a light microscope using a 100× objective lens (Axo observer 3; Zeiss, Oberkochen, Germany). To investigate the localization of the Cdh1 protein under different stress conditions, we inoculated the GFP-Cdh1 fluorescent strain into a YPD medium containing various stresses such as cell integrity stress (0.025% SDS and 0.5% Congo red), osmotic stress (1.5 M NaCl and 1.5 M sorbitol), and oxidative stress (2.5 mM H₂O₂). Subsequently, the subcellular localization was observed with fluorescence microscopy (Axo observer 3; Zeiss, Oberkochen, Germany) after 24 h.

2.4. Assays of Melanin and Capsule Production and Growth Under Stress Conditions

To assess melanin production in C. neoformans strains, 100 μL of each overnight culture was inoculated onto a Niger seed agar medium. The agar plates were then incubated at either 30 °C or 37 °C for 2 days, after which the pigmentation of the fungal colonies was evaluated and photographed for recording. For capsule production analysis, overnight cultures were washed three times with phosphate-buffered saline (PBS) and resuspended at a concentration of 1 × 10⁶ cells/mL in a DME medium. The cells were incubated in a 10% CO₂ atmosphere for 24 h. After the capsule induction, the cells were mixed with India ink, observed by a light microscope using a 100× objective lens (Axo observer 3; Zeiss, Oberkochen, Germany), and photographed for recording. The capsule length of C. neoformans ((extracellular diameter-intracellular diameter)/2) was measured using Image J, and at least 100 cells were measured for each strain.

To investigate the role of Cdh1 in the stress resistance of C. neoformans, we examined the growth of each cryptococcal strain on YPD plates containing different stressors. Overnight cultures of each C. neoformans strain were collected, washed three times in sterile PBS, and diluted to an OD₆₀₀ of 2.0. The yeast cells were serially diluted ten-fold in ddH₂O, and 5 μL aliquots of each dilution were plated. The plates were incubated at 30 °C for 2–3 days, and the growth status of the strains was recorded by photographing.

To evaluate the role of Cdh1 in the growth of Cryptococcus under different temperatures, we adjusted the initial concentration of the inoculum to 1 × 10⁷ cells/mL and incubated 100 μL of the inoculum at 30 °C and 37 °C for 36 h, with measurements taken every 2 h. The optical density (OD₆₀₀) was determined using the BMG LABTECH multifunctional microplate reader (FLUOstar Omega, Ortenberg, Germany).

2.5. Virulence Studies

In this study, overnight cultures of each cryptococcal strain were washed three times with 1 × PBS buffer and then resuspended at 2 × 10⁶ cells/mL in 1 × PBS buffer. Subsequently, ten female eight-week-old C57 BL/6 mice (Hunan SJA Laboratory Animal, Changsha, China) were intranasally inoculated with 50 μL of cell suspension of each strain, resulting in an inoculum of 1 × 10⁵ cells per mouse. The infected mice were regularly monitored after inoculation, and their survival times were recorded. The survival curve of the mice was then plotted using GraphPad Prism 8.0.

To further investigate the role of Cdh1 in the pathogenicity of C. neoformans, we conducted an assessment of the virulence associated with Cdh1-related strains using the Wax moth larva (Galleria mellonella) model, as outlined in previously published protocols [46]. Overnight cultures of each strain were harvested, subjected to two washes with sterile 1 × PBS buffer, and then adjusted to a concentration of 1 × 10⁸ cells/mL in 1 × PBS buffer. Groups of 10 larvae, with an average body weight of 300 mg, were inoculated with 10 μL of the inoculums with a microliter syringe, targeting the area of the last pro-leg. Prior to injection, the pro-leg area was disinfected with 70% ethanol using a swab. Following inoculation, the larvae were placed in 90 mm plastic Petri dishes and incubated at 30 °C and 37 °C in the dark for 16 days. During the incubation period, the larvae were monitored daily by observing spontaneous or provoked movements, facilitated by the use of a previously sterilized clip. The larvae were deemed deceased when they exhibited a lack of response to gentle prodding with forceps. Survival curves were generated, and the statistical analysis of survival differences was conducted employing the Kaplan–Meier method, utilizing GraphPad Prism 8.0.

All animal studies conducted at Southwest University were according to the “Guidelines on Ethical Treatment of Experimental Animals (2006, No. 398)” and the “Regulations for the Management of Laboratory Animals” (2006, No. 195) issued by the Ministry of Science and Technology of China. Furthermore, the Animal Ethics Committee at Southwest University approved all of the vertebrate studies under Protocol No. IACUC-20221022-14).

2.6. Histopathology and Fungal Burdens of Infected Tissues

The infected lungs, brains, and spleens of the mice were dissected, and half of the organs were fixed with 10% formalin for histopathological section preparation by Servicebio (Servicebio, Wuhan, China). The other half of the organs were homogenized in 3 mL of sterile 1 × PBS buffer, and 100 μL of the serial dilutions of the homogenates were plated on YPD agar plates containing 50 mg/L chloramphenicol and 50 mg/L kanamycin, followed by an incubation period of 2–3 days at 30 °C. The resulting colonies were counted, and the tissue fungal burdens were calculated as CFU/g tissue weight, represented as log10. Additionally, the histopathological section of infected tissues was stained with hematoxylin and eosin (H&E) and recorded using a light microscope with a 40× objective lens (Axo observer 3; Zeiss, Oberkochen, Germany).

2.7. Cryptococcus–Macrophage Interaction Assay

Macrophage-like J774 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C under a 5% CO₂ atmosphere. A total of 5 × 10⁴ J774 cells in 0.5 mL of fresh DMEM were seeded into each well of a 48-well culture plate and incubated overnight at 37 °C in 5% CO₂. To facilitate the activation of macrophage cells, 50 units/mL of gamma interferon (IFN-γ; Invitrogen) and 1 µg/mL of lipopolysaccharide (LPS; Sigma) were administered to each well. C. neoformans overnight cultures were subsequently washed twice with phosphate-buffered saline (PBS) and opsonized using 10 µg/mL 18B7 mAb (Meridian Bioscience, Beijing, China). A total of 2 × 10⁵ C. neoformans cells were introduced to each well, achieving a yeast-to-J774 cell ratio of 4:1. To evaluate the intracellular proliferation of C. neoformans, non-adherent extracellular yeast cells were removed by washing with fresh DMEM following 2 h of co-incubation. The cultures were further incubated for additional time points of 0, 2, or 22 h. At these specified time intervals, the medium in each well was replaced with distilled water (dH₂O) to lyse the macrophage cells for 30 min at room temperature. The resulting lysate was then spread onto YPD plates containing 50 mg/L chloramphenicol and 50 mg/L kanamycin and incubated at 30 °C for 2 days, after which colony-forming units (CFU) were counted to assess the intracellular proliferation of the organism.

Serum treatment and the C. neoformans cell viability assay were conducted as previously described [47]. At the specified time points, aliquots were removed and plated onto a YPD medium after serial dilution to assess cell viability.

2.8. Statistical Analysis

Statistical analysis and plotting of the data in this study were conducted using Prism 8.0. Significance was determined using a one-way ANOVA with Tukey’s multiple comparisons in GraphPad Prism. A log-rank test was utilized to analyze the differences in pathogenicity between mice or between the Wax moth larvae infected with different strains. All data are presented as mean ± standard deviation, and a p-value < 0.05 was considered statistically significant.

3. Results

3.1. Identification of the Anaphase-Promoting Complex Activator Cdh1 in C. neoformans

In our previous study, we identified the potential RNA targets associated with the RNA-binding protein Grp1. Our findings suggest that CNAG_03191 is a promising candidate gene associated with Grp1(data not published). We downloaded the sequence information of CNAG_03191 from the Fungi DB website (https://fungidb.org/fungidb/app/record/gene/CNAG_03191, accessed on 30 June 2022) and conducted a thorough analysis. The sequence analysis revealed that the full length of CNAG_03191 was 2726 bp, containing four exons and three introns (Figure 1A). Furthermore, domain analysis showed that CNAG_03191 contained six WD40 repeats (Figure 1B). The WD40 repeat is a short ~40 amino acid motif, usually terminated by a Trp-Asp (W-D) dipeptide, which is widespread and highly conserved in eukaryotes and usually folds into a β-propeller structure [48]. We searched for sequences of S. cerevisiae with high similarity to C. neoformans CNAG_03191 on NCBI and then compared and analyzed these sequences using the clustal W (https://www.genome.jp/tools-bin/clustalw/, accessed on 30 June 2022) and ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/, accessed on 30 June 2022) tools. Our results demonstrated that the amino acid sequence of C. neoformans CNAG_03191 shares 73.38% similarity (Figure 1C) with the S. cerevisiae anaphase-promoting complex activator Cdh1; so, we designated CNAG_03191 as Cdh1 based on this similarity.

3.2. Cdh1 Is Localized in the Nucleus of C. neoformans

To determine the subcellular localization of Cdh1 protein in C. neoformans, a GFP-CDH1 fusion expression vector, pTBL435, was constructed and transformed into the cdh1Δ mutants using biolistic transformation. Following resistance screening and fluorescence microscopy observation, the GFP-Cdh1 green fluorescent strain was obtained. The fluorescence observation revealed that the GFP-Cdh1 fusion protein exhibited a punctate green fluorescence signal and was situated at the center of C. neoformans cells. Further confirmation of the localization of Cdh1 was achieved by staining cells with DAPI, which demonstrated the co-localization of Cdh1 with DAPI, indicating its presence in the nucleus (Figure 2A). Moreover, the expression of the GFP-Cdh1 fusion protein remained stable in the nucleus of C. neoformans under various stress conditions (Figure 2B).

3.3. Construction of Cdh1-Related Strains

To investigate the functional role of Cdh1 in C. neoformans, we constructed Cdh1-related strains. The target gene CDH1 was replaced with the NEO marker gene using a Split Marker strategy. Subsequently, seven cdh1Δ mutant strains were obtained and confirmed via PCR and Southern blot. The plasmid for CDH1 gene complementation, pTBL404, was transformed into the cdh1Δ mutant strain using biolistic transformation. Additionally, to obtain the CDH1^OE overexpression strain, i.e., the constructed CDH1^OE overexpression vector pTBL443 in which the expression of the CDH1 controlled by actin promoter was transformed into the cdh1Δ mutant strain by biolistic transformation. The expression of the CDH1 gene in transformants was confirmed through resistance screening, Western blot, and qRT-PCR analysis. As shown in Figure 3A,B, the Cdh1-HA protein size was consistent with expectations, and the RNA expression level was significantly higher compared to that of the wild-type H99 strain, thus successfully yielding the CDH1^OE overexpression strain.

3.4. Cdh1 Affects the Production of Virulence Factors of C. neoformans

The ability to grow at 37 °C, form melanin, and produce a polysaccharide capsule represent the three main virulence factors of C. neoformans, crucial for its survival in infected hosts and the initiation of cryptococcosis. To investigate the role of Cdh1 in the production of these virulence factors, we incubated cdh1Δ mutant, cdh1Δ::CDH1 complementary, and CDH1^OE overexpression strains at 37 °C, plated them on a Niger seed medium to induce melanin formation, and inoculated them in a SAB + MOPS medium to induce capsule growth in order to observe the formation of virulence factors. The results of the capsule formation of the strain are shown in Figure 3C,D. Compared to the wild-type strain H99, the capsule thickness of the cdh1Δ mutant strain was significantly reduced. Similar results were observed with the overexpression of CDH1, indicating the importance of Cdh1 in the capsule synthesis of C. neoformans and its involvement in the process of capsule formation. Additionally, we examined the effects of the cdh1Δ mutant and CDH1^OE overexpression strains on melanin formation, and the results showed that the deletion and overexpression of the CDH1 gene did not affect melanin formation in C. neoformans (Figure 3C).

To evaluate the role of Cdh1 on the growth of C. neoformans under different stress conditions, the series-diluted strains were then plated on YPD agar plates and incubated at 37 °C for two days for observation. After incubation, we found that the cdh1Δ mutant strain grew slowly at 37 °C (Figure 3E), indicating that Cdh1 is involved in maintaining the growth of C. neoformans at 37 °C. Meanwhile, we also noticed a slight growth defect of the cdh1Δ mutant in YPD with 1.5M KCl or 1.5M NaCl, indicating that Cdh1 is important for C. neoformans to respond to high-salt environments. Concurrently, we assessed the growth of the cdh1Δ mutant in a liquid YPD medium. The results revealed that the growth rate of the cdh1Δ mutant at 30 °C was comparable to that of other strains. However (Figure 3F), at 37 °C, its growth rate was significantly diminished relative to the other strains (Figure 3G). These findings suggest that the disruption of the CDH1 gene affects capsule formation and growth at 37 °C or in high-salt environments, thereby indicating a potential role for Cdh1 in the pathogenicity of C. neoformans.

3.5. Cdh1 Regulates Fungal Virulence in C. neoformans

To explore the effect of Cdh1 on fungal virulence, C57 BL/6 mice were intranasally infected with the strains to assess the role of Cdh1 in the virulence of C. neoformans. Statistical analysis revealed that the mice infected with the wild-type H99 strain succumbed to the infection 19 days post-infection(dpi), with all of them dying within 28 days. In contrast, mice infected with the cdh1Δ mutant strain began to die at 30 dpi and did not all succumb until 80 dpi, signifying a significant decrease in pathogenicity. When infected with the cdh1Δ::CDH1 complemented strain, the pathogenicity of the strain was not significantly different from that of the wild-type H99 strain. The mice began to die at 21 dpi, with all deaths occurring within 26 days. Following infection with the CDH1^OE overexpression strain, the mice started to die at 37 dpi, and all died within 55 days (Figure 4A).

To understand why the cdh1Δ mutant and CDH1^OE overexpression strains showed decreased virulence, we counted the fungal burden in the infected mice at the end of the animal experiment. The results showed no significant difference in the CFU statistics of different tissues of mice infected with cdh1Δ mutant, cdh1Δ::CDH1 complemented, and CDH1^OE overexpression strains compared to mice inoculated with the H99 strain (Figure 4B). Pathological tissue sections revealed that infection with cdh1Δ mutant, cdh1Δ::CDH1 complemented, and CDH1^OE overexpression strains resulted in severe damage to the main infected organs of mice, including lungs, brains, and spleens, with noticeable tissue lesions and a large number of yeast cells (Figure 4C, black arrow). This result was consistent with the tissue CFU statistics. Based on the fungal burden statistics and pathological section results of dead mice at the end of the experiment, it was evident that C. neoformans could accumulate in large quantities in the body after infection with cdh1Δ mutant and CDH1^OE overexpression strains, ultimately leading to the death of the mice. However, it was observed from the survival curves that infection with the cdh1Δ mutant and the CDH1^OE overexpression strains prolonged the survival days of mice and significantly decreased pathogenicity.

The markedly reduced growth rate observed in the cdh1Δ mutant compared to the wild-type strain at 37 °C indicates that the diminished pathogenicity of the cdh1Δ mutant during in vivo infection may be primarily attributable to its impaired growth at 37 °C. This finding complicates the effort to delineate the loss of virulence from thermosensitive effects. To further investigate this phenomenon, we conducted a pathogenicity assay utilizing a wax moth larvae model. The results demonstrated a significant attenuation of virulence in the cdh1Δ mutant at both 30 °C (Figure 4D) and 37 °C (Figure 4E), suggesting that the observed loss of pathogenicity in the cdh1Δ mutant is not merely a consequence of reduced heat resistance.

3.6. Cdh1 Regulates the Progression of Fungal Infection

The function of Cdh1 in C. neoformans infection was investigated through the quantification of fungal burden and histopathological analysis of lung, brain, and spleen tissues at 7, 14, and 21 dpi with the H99 strain, cdh1Δ mutant, and CDH1^OE strains. In this study, we closely monitored the dynamic changes of C. neoformans within the host. Tissue fungal burden analysis indicated that yeast cells of C. neoformans were recovered from the tissues of mice infected with the H99 strain at 7 dpi, with the fungal burden progressively increasing over time (Figure 5A). Mice infected with the cdh1Δ mutant and CDH1^OE strains exhibited a similar trend in lung tissue but with a significantly lower fungal burden than the H99 strain. At 21 dpi, the cdh1Δ mutant and CDH1^OE strains began to spread to the brains, while no cryptococcal cells were isolated from the spleens. Histopathological sections revealed the presence of yeast cells in lung tissues at 7 dpi in all infected groups, with tissue damage increasing over time. However, infection with the cdh1Δ mutant strain or the CDH1^OE strain caused less tissue damage than the H99 strain (Figure 5B, left panel). At 21 dpi, lesions and a large number of yeast cells of C. neoformans were detected in the brains or spleens of mice infected with the H99 strain, whereas no significant lesions or yeast cells were found in the brains and spleens of mice infected with the cdh1Δ mutant and CDH1^OE strains until 21 dpi (Figure 5B, middle and right panels), indicating that the progression of the fungal infection is significantly delayed in mice infected with the cdh1Δ mutant and CDH1^OE strains. The findings from tissue sampling at different time points underscore the critical role of Cdh1 in the progression of C. neoformans infection (Figure 5D).

3.7. Cdh1 Affects the Proliferation of C. neoformans Inside Macrophages

The results of the fungal pathogenicity assay showed that Cdh1 affected the virulence of C. neoformans. To elucidate the reasons for the changes in the pathogenicity of cdh1Δ mutant and CDH1^OE strains in the host, the growth dynamics of C. neoformans cells after incubation with mouse serum for varying durations were examined to investigate the potential influence of host serum components on the growth of C. neoformans. The wild-type strain H99 served as the control, while the cdh1Δ mutant and CDH1^OE strains were co-incubated with mouse serum. Subsequently, the growth of each strain was assessed after 1, 2, 3, and 4 h of co-incubation. The findings showed that there was no significant difference in survival rates among the cdh1Δ mutant, CDH1^OE, and wild-type strains after co-incubation with mouse serum. This suggests that host serum components did not exert a discernible impact on the growth of Cdh1-related strains (Figure 6A).

The pulmonary macrophages serve as the first line of defense against C. neoformans. Therefore, we investigated how CDH1-related strains interact with J774 macrophages from mice. There were no significant differences between the cdh1∆ mutant, CDH1^OE strain, and the wild-type H99 strain after 2 and 4 h of co-incubation with macrophages. However, after 24 h of incubation, the proliferation of the cdh1∆ mutant and CDH1^OE strains did not increase as much as the wild-type H99 strain and the cdh1Δ:: CDH1 complemented strain but instead remained at a proliferation level comparable to that observed after 4 h of incubation (Figure 6B). These findings indicate that the reduced pathogenicity of the cdh1Δ mutant and CDH1^OE strains results from their limited proliferation within host macrophages.

3.8. Dynamic Changes of Chemokines in Lung Tissues of Mice Infected with Cryptococcus Strains

Chemokines are crucial in generating adaptive immune responses and promoting the pathogenesis of various diseases. Compared to the H99 strain infection, Cxcl1, which can recruit and activate neutrophils, and Ccl17, secreted by M2 macrophages to act on Th2 cells, showed a gradually increasing expression level in the lungs of mice infected with the cdh1Δ mutant strain. Notably, after 14 and 21 days of infection, the expression levels of CXCL1 and CCL17 significantly increased compared to the H99 infection group (Figure 7). Conversely, following infection with the CDH1^OE strain, the expression of CXCL1 in mouse lungs exhibited a gradual decline, ultimately reaching levels that were significantly lower than those observed in the H99 infection group by day 21. Furthermore, Ccl20, which plays a crucial role in the migration of Th17 cells to the site of inflammation, showed no significant difference in expression in the lungs of mice infected with the cdh1Δ mutant strain 7 and 14 dpi. However, a marked increase in the expression of CCL20 was noted 21 dpi with the cdh1Δ mutant strain (Figure 7). In the early stage of infection (7 dpi), both the cdh1Δ mutant and CDH1^OE strain infections were shown to stimulate lung tissue to secrete greater levels of the Th1 chemokine Cxcl10, which is known for its direct killing activity against C. neoformans [49]. Nevertheless, the expression level of CXCL10 exhibited a significant reduction in subsequent stages (14 and 21 dpi) (Figure 7). Moreover, Ccl2, a chemokine critical for the promotion of monocyte/macrophage migration and infiltration to infection sites, displayed significantly lower expression levels in the lungs of mice infected with cdh1Δ mutant and CDH1^OE strains 14 and 21 dpi, in contrast to the H99 group. The expression levels of CXCL1, CCL17, and CCL20 were significantly upregulated 21 days after infection with the cdh1Δ mutant strain, indicating a pronounced activation of lung tissue recruitment of neutrophils and enhanced Th2 and Th17 immune responses (Figure 7). Additionally, the elevated expression of CCL20 across all time points following CDH1^OE strain infection suggests that the Th17 immune response is more robustly activated in this context. The observed low levels of CCL2 imply that infections with the cdh1Δ mutant and CDH1^OE strains exert a limited effect on macrophage activation, resulting in fewer monocytes/macrophages migrating to the lungs.

4. Discussion

C. neoformans is a critical fungal pathogen that threatens public health worldwide. Due to the limited availability of antifungal drugs and the emergence of drug-resistant strains with toxic side effects, it is crucial to explore effective treatments for cryptococcosis. Researching the pathogenesis of C. neoformans is important for better understanding the fungus, identifying new antifungal drug targets, and developing more effective antifungal drugs. To investigate the role of the Cdh1 protein in the pathogenicity of C. neoformans, we created the CDH1 gene disruption, complementation, overexpression, and GFP-Cdh1 fusion expression strains using biolistic transformation. Subcellular localization studies revealed that the Cdh1 protein is located in the nucleus of C. neoformans. The cdh1Δ mutant strain showed a growth defect at 37 °C. Both CDH1 deletion and overexpression significantly reduced the capsule thickness of C. neoformans, suggesting that Cdh1 may play a role in regulating capsule formation. Furthermore, our results from the murine inhalation model of the systemic infection of cryptococcosis also indicate that Cdh1 plays a significant role in the pathogenicity of C. neoformans.

In C. neoformans, CNAG_03191 was annotated as a putative protein. Since its sequence has the highest sequence similarity as that of S. cerevisiae Cdc20 homolog 1 (Cdh1) and both contain the WD40 domain, it was also named Cdh1 in this paper. Repetitive WD domain proteins are a large family in all eukaryotes and are involved in various processes, such as signal transduction, transcriptional regulation, cell cycle control, and apoptosis [48]. In mitotic cells, the activity of the Cdh1 protein shows cellular periodicity; Cdh1 maintains a low phosphorylation level in the G1 phase and mitosis anaphase and a high phosphorylation level in the G2 phase, the S phase, and early mitosis [50]. Cdh1 is maintained in a repressive state at mitosis telophase and in the G1 phase to ensure that cells undergo precise DNA replication during the S phase. In the late stage of mitosis, the anaphase-promoting complex (APC) can be activated by cell division cycle 20 homolog (Cdc20) or Cdh1, which degrades cell cycle proteins through ubiquitination, thereby inactivating the corresponding CDK and completing a cell cycle [51]. In vertebrates, the loss of Cdh1 results in the accumulation of mitotic cell cycle proteins cyclin A and cyclin B in the G1 phase and a delay in mitotic exit [50]. As the role of Cdh1 in the cell cycle of C. neoformans is still unknown, the function of the CDH1 gene in C. neoformans can be fully elucidated by examining the cell cycle progression of the cdh1∆ mutant strain.

In budding yeast, Cdh1 mediates the degradation of APC/C substrates, and the Cdc20/Cdh1 protein family can be used as specific activators of downstream substrates of APC/C [52,53]. This conclusion was also confirmed by Gieffers et al., who proposed that Cdh1 is the activating subunit of APC/C [54]. Cdc20 and Cdh1 function at different cell cycle stages and contribute to the substrate specificity of APC/C. Cdc20 activates APC in early mitosis, while Cdh1 activates APC in G1 and mitosis anaphase [55]. As a target gene of Grp1, the downstream substrate of F-box protein Fbp1 (data not published), whether Cdh1 also has the function of specifically activating Grp1, the downstream substrate of the E3 ubiquitin ligase SCF complex, remains to be further explored.

The capsule is the main virulence factor, which can inhibit the phagocytic activity of host immune cells towards C. neoformans. Mutant strains with a damaged capsule structure are often more susceptible to phagocytosis by macrophages [10,11,12], and an increase in thickness reduces phagocytic efficiency, making it difficult for cells to break through the blood–brain barrier [8,9], indicating that the normal formation of capsules is closely related to virulence. The deletion of the CDH1 gene significantly reduced the capsule thickness of C. neoformans, and overexpression of this gene also showed similar results (Figure 3C,D), indicating that the normal expression of CDH1 plays an important role in capsule synthesis and may be involved in regulating the process of capsule formation. The performance of Cdh1 in capsule synthesis and growth at 37 °C also indirectly suggests its possible association with pathogenicity.

The intranasal inhalation experiment conducted in mice demonstrated that infection with the cdh1Δ mutant and CDH1^OE strain led to a notable extension in the survival time of the mice and a reduction in the pathogenicity of the strains (Figure 4A). In addition, the assessment of tissue fungal burden and observation of tissue lesions at specific time points for mice inoculated with cdh1Δ mutant and CDH1^OE strains revealed a gradual increase in the number of C. neoformans in tissues throughout the infection, albeit at significantly lower levels compared to the wild-type strain (Figure 5A). The infection kinetics of the cdh1Δ mutant and CDH1^OE strains were found to be akin to those of the wild-type H99 strain, although they were significantly delayed. The growth rate of the cdh1Δ mutant was observed to be significantly lower than that of the H99 strain at 37 °C, as illustrated in Figure 3G. Together with the pathogenicity assay outcomes conducted using Galleria mellonella larvae, depicted in Figure 4D,E, these findings suggest that the diminished pathogenicity of the cdh1Δ mutant strain primarily arises from impaired thermotolerance. Conversely, the CDH1^OE strain presents a more intricate mechanism of action, exhibiting normal growth at 37 °C while simultaneously demonstrating a pronounced reduction in virulence. This phenomenon necessitates further investigation to elucidate the underlying mechanisms.

Human respiratory tract infection occurs by inhaling dried spores or yeast cells of C. neoformans in the environment. Host immune cells, including macrophages and dendritic cells, are the first to interact with invading pathogens and participate in the early recognition and clearance of infection [56]. Here, the interaction between C. neoformans and macrophages was examined to explore the reasons for the changes in the pathogenicity of Cdh1-related strains in the host. When the Cdh1-related strains were co-incubated with mice macrophages, the cdh1∆ mutant strain showed a significant decrease in proliferation at 24 h of incubation (Figure 6B), which was consistent with the slower growth of the strain at 37 °C when the strain was tested for virulence factors (Figure 3E), indicating that the growth of the strain itself caused the reduced proliferation of the cdh1∆ mutant strain in macrophages.

Exploring the immune response during the interaction between hosts and pathogens can help us better understand the molecular mechanisms of pathogen invasion in hosts and provide ideas for analyzing pathogenicity and finding therapeutic targets. According to qRT-PCR analysis of chemotactic factors in mice lung tissue, infection with the cdh1Δ mutant strain can activate the recruitment of neutrophils in lung tissue, as well as Th2 and Th17 immune responses, to a greater extent. At the same time, its effect on macrophage activation is limited (Figure 7). The chemokine Cxcl1 recruits and activates neutrophils to combat infections and kills microorganisms in tissues [57,58]. Although neutrophils contribute to resistance to C. neoformans infection, it is unnecessary for the host to clear C. neoformans [59,60]. Cxcl10, belonging to Th1 chemokines, can target the cell wall/membrane and intracellular structures of C. neoformans and have direct killing activity against C. neoformans in vitro [49]. In the early stage of infection (7d), infection with the cdh1Δ mutant strain can stimulate more robust secretion of Cxcl10 in lung tissue, but in the later stage, the expression level of CXCL10 is significantly reduced, which may be the reason why infection with the cdh1Δ mutant strain cannot effectively be eliminated from the body of the mice.

Currently, antifungal drugs used to treat cryptococcosis include azoles, polyenes, and pyrimidine analogs. The azoles specifically target the ergosterol biosynthetic enzyme Erg11, while polyenes directly target ergosterol, and pyrimidine analogs block the synthesis of DNA and RNA [61]. In the future, we can explore the sensitivity of the cdh1Δ mutant strain to various antifungal drugs, which may lead to the discovery of new therapeutic targets and facilitate the development of improved antifungal medications.

As a basidiomycete with a bipolar mating system, C. neoformans has a chromosomal region with a mating type locus, the MAT locus, which corresponds to two mating types: α and a. Strains with different mating types can carry out cell fusion, hyphal growth, and basidiospore production under mating induction conditions [62,63]. Basidiospores produced through the sexual reproduction process are resistant to external stress environments and are important pathogens that can cause fatal cryptococcal meningitis [64]. Wu et al. showed that the deletion of CDC4, a cell cyclin-related protein, could not undergo nuclear division during mating, blocking meiosis, thus affecting the sexual reproduction of C. neoformans [65]. The mutant strain lacking the cell cycle-related protein Cbc1 lost the ability to produce basidiospores [33]. These results suggest that the cell cycle-related proteins of C. neoformans play an essential role in producing infectious basidiospores. In this study, we mainly explored the effect of Cdh1 on the pathogenicity of C. neoformans but did not study its role in sexual reproduction. In the future, the molecular mechanism of Cdh1 in the mating sporulation can be further analyzed to provide biological data for the morphological development of C. neoformans.

Author Contributions

Conceptualization, T.-B.L. and Q.-H.L.; methodology, Q.-H.L.; software, L.-T.H.; validation, Q.-H.L., L.-T.H., and M.-R.G.; formal analysis, Q.-H.L.; investigation, Q.-H.L.; resources, Q.-H.L.; data curation, Q.-H.L.; writing—original draft preparation, Q.-H.L.; writing—review and editing, T.-B.L.; visualization, Q.-H.L.; supervision, T.-B.L.; project administration, C.-L.F.; and funding acquisition, T.-B.L. and C.-L.F. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Southwest University (protocol code IACUC-2022-14, 22 October 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10120891/s1, Table S1: Strains used in this study; Table S2: Plasmids used in this study; and Table S3: Primers used in this study.

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