All experiments were conducted using the wildtype C. neoformans strain, H99S (serotype A) (Perfect et al., 1980; Janbon et al., 2014), unless otherwise stated. H99S was grown from frozen stock in yeast peptone dextrose (YPD) broth (Fisher Scientific; Hampton, NH, United States) for 24–36 h (log-phase) in an orbital incubator at 37°C for all treatments. After incubation, the cells were washed in phosphate-buffered saline (PBS) three times and resuspended in PBS. Cells were counted and diluted to a final concentration of 1 × 10⁶ cells/mL. Clinical isolates obtained from the cerebrospinal fluid of HIV⁺ patients with cryptococcal meningitis in Botswana (Bisson et al., 2008) were used in initial experiments to determine if melanization varied from the wildtype. C. neoformans knockout (KO) strains of genes identified from the RNA sequencing data were a kind gift from Dr. Hiten Madhani (University of California, San Francisco) and did not show a melanization phenotype (Liu et al., 2008).

H99S and clinical isolates were grown and prepared as stated above. Using a modified melanization protocol, exogenous hormones were added at physiological concentrations (17β-estradiol: 400 pg/mL (Calbiochem, San Diego, CA, United States; testosterone: 10 ng/mL, Sigma Aldrich, St. Louis, MO, United States) to the 1 mM L-DOPA (0.1982 g, Sigma Aldrich, St. Louis, MO, United States) liquid minimal media and agar plates (Eisenman and Casadevall, 2012). Ethanol was used as the diluent for both steroid hormones and was therefore used as a vehicle control. Three clinical strains and H99S (1 × 10⁶ cells per strain) were streaked onto each plate (one per quadrant, see Figure 1A). The plates and flasks were wrapped in foil for protection from light and stored in a 37°C incubator for 5 days (liquid media) or 7 days (plates). One milliliter was removed from the flasks every 24 h for an absorbance measurement. An optical density of 400 and 600 nm (to correct for cell density) was determined as by Baker et al. (2007). On days three, five, and seven, images were taken of the L-DOPA plates to document the amount of melanization.

Conditioned media was made using the L-DOPA liquid media experiment described above. C. neoformans was grown in liquid L-DOPA media supplemented with steroid hormones for 7 days to allow melanization. On day seven, melanized cultures were centrifuged at 750 × g for 5 min to pellet cells and supernatants were collected. Supernatants were plated to determine that all cells from the culture were removed. A fresh C. neoformans culture was started on day six and prepared as stated above. In three flasks treated with physiological concentrations of testosterone, estrogen, or a 1:100 dilution of ethanol, respectively, new L-DOPA media and the collected supernatants of the melanized cultures were added in a 1:1 fashion with fresh C. neoformans cells. The flasks were wrapped in foil for protection from light and incubated at 37°C in an orbital shaker at 150 RPM. Absorbance measurements at an optical density of 400 and 600 nm were taken at three timepoints, 16, 24, and 32 h, and the pH was tested for each hormone treatment using pH tests strips.

H99S was grown to log-phase, as stated above. Five concentrations of exogenous testosterone (0, 0.5, 2, 5, and 10 ng/mL) and estradiol (0, 100, 200, 400, and 10 ng/mL) were added to the cells and conditioned L-DOPA media, respectively. The flasks were protected from light and incubated for 32 h in a 37°C orbital shaker at 150 RPM. An absorbance measurement at an optical density of 400 and 600 nm was taken at three timepoints, 16, 24, and 32 h.

H99S was grown to log-phase, as stated above. Three flasks were treated with testosterone, estrogen, or ethanol, respectively. Cells, hormones, and L-DOPA liquid media were all added to the flasks at a final volume of 10 mL. The flasks were protected from light and placed in a 37°C orbital shaker for 3 days. After incubation, cells were centrifuged at 750 × g for 5 min, counted, and diluted to a final concentration of 2 × 10⁸ cells for RNA isolation using an RNeasy Midi Kit (Qiagen, Hilden, Germany). Genomic DNA contamination was removed from the total RNA using a Message Clean Kit (GeneHunter, Nashville, TN, United States), and the RNA concentration was measured using a NanoDrop 2000 Spectrophotometer (Thermo-Scientific, Waltham, MA, United States).

qRT-PCR was performed using RNA isolated from C. neoformans at day three of melanization. cDNA was prepared using the QuantiNova^TM Reverse Transcription kit (Qiagen, Hilden, Germany) using 1 μg of isolated RNA. cDNA, primers, and SYBR green were added to a 96-well PCR plate that amplified the products using a CFX96 Real-time PCR Detection System (BioRad Laboratories, Hercules, CA, United States). Each cDNA treatment was performed in quadruplicate and normalized using the geometric mean of the housekeeping genes UBC6 and TFC1. Fold changes were determined using the 2^–ΔΔC′T method, as described by Livak and Schmittgen (2001).

cDNA libraries for RNA sequencing (RNAseq) were prepared from 1 μg of isolated RNA using the NEBNext Ultra^TM Directional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, United States), the NEBNext Multiplex Oligos for Illumina Index Primers and the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs, Ipswich, MA, United States). Sequencing (75 bp, paired end with 22–57 million reads) was completed by HudsonAlpha Genomic Sequencing Lab (Huntsville, AL, United States) using the HiSeq 2500 system.

RNAseq analysis was completed using two analytical platforms, CyVerse and Galaxy. Quality check was completed on each paired-end read using FASTQC (version 0.11.5 multi-file). Due to issues with STAR (version 2.5.3a-index-align), the paired-end reads were aligned using HISAT2 (version 2.1-index-align) to a C. neoformans (H99S) reference genome found using ENSEMBL.

Bam files produced from the HISAT2 (version 2.1-index-align) alignment were utilized in two ways. In Galaxy, the bam files were used to quantify the number of reads using a genome annotation file of C. neoformans found in ENSEMBL. MultiJoin (also in Galaxy) was used to join the output files of featureCounts into a single tabular file. This file was utilized in R studio using the edgeR differential expression package. To determine the similarities among replicates in the treatment groups (testosterone, estrogen, and ethanol) and to ensure any outliers would be removed, multidimensional scale plots (MDS plot) and a cluster dendrogram were utilized.

Once cluster analysis was complete, the reads were aligned into any potential transcripts using StringTie and the genome annotation (version 1.3.3) (CyVerse). Transcripts for all replicates in the treatment groups were then merged into a single gtf file using StringTie-Merge (version 1.3.3). Lastly, CuffDiff2 (version 16-way max) was utilized to compare the genome alignments (bam files) and the transcript alignments (gtf files) for gene expression changes that were significantly different. This output file was used to determine any genes that were upregulated when C. neoformans was in the presence of testosterone compared to the other treatment groups.

GA (Sigma Aldrich, St. Louis, MO, United States) and PA (MP Biomedicals, Santa Ana, CA, United States) concentrations in C. neoformans supernatants during melanization were determined using ELISAs. A standard curve for both PA (9.76–1250 ng/mL) and GA (62.5–500 ng/mL) were utilized (Gonthier et al., 1998; Gokani and Thaker, 2002). A primary rabbit polyclonal antibody (Lifespan Biosciences, Seattle, WA, United States) for both GA (1/400 dilution) and PA (1/600 dilution) was added to the samples, and they were incubated in a 37°C water bath for 1 h. After incubation the samples were transferred to a 96 well flat-bottom microplate (Greiner Bio-One, Kremsmunster, Austria) in duplicate and incubated again at 37°C for 1 h. Using a microplate washer (BioTek, Winoski, VT, United States), the wells were washed with PBS + 0.5% Tween 20 wash buffer. A secondary donkey anti-rabbit horseradish peroxidase polyclonal antibody (1/4000 dilution, Southern Biotech, Birmingham, AL, United States) was added to each well and the plate was incubated for another hour at 37°C. After incubation, the plate was washed using PBS + 0.5% Tween 20. The 3,3′,5,5′-tetramethylbenzidine substrate (TMB, Thermo Fisher Scientific, Waltham, MA, United States) was added to each well, and the plate was incubated for 30 min at room temperature. After incubation, 2 N sulfuric acid stop solution was added to each well. The absorbance of each sample was measured using the CLARIOstar Plus Microplate Reader (BMG LABTECH, Offenburg, Germany) at 450 nm.

KO strains (a kind gift of Dr. Hiten Madhani, UCSF) of C. neoformans genes identified in the RNA sequencing and H99S were prepared as stated above. Following similar methods of the initial L-DOPA liquid media experiments (above), exogenous testosterone was added to all flasks containing strains and the flasks were incubated for 5 days. One milliliter was removed from the flasks every 24 h for an absorbance measurement at an optical density of 400 and 600 nm.

H99S cells were prepared as stated above, with the exception of a starting cell concentration of 1 × 10⁵ cells/mL. Following similar methods of the initial L-DOPA liquid media experiments, exogenous steroid hormones were added to all flasks containing H99S and incubated in a 37°C orbital shaker at 150 RPM for 9 h. After the 9 h incubation, one milliliter was removed from the flasks every 6 h for an absorbance measurement at an optical density of 600 nm. Measurements continued until all cultures reached an absorbance of 1.00 or stopped growing.

To test the effects of PA and GA on melanization, the conditioned media experiment (above) was modified. Using snap-cap tubes, a variety of additives/inhibitors were added to tubes containing steroid hormone supplemented conditioned L-DOPA media and C. neoformans cells. The additives/inhibitors included: 3 μg/mL GA (Sigma Aldrich, St. Louis, MO, United States), 2 × 10^–6 M chlormequat chloride (GA inhibitor, Sigma Aldrich, St. Louis, MO, United States), 10 μM PA (MP Biomedicals, Santa Ana, CA, United States), and 1 mg/mL DL-Serine (PA inhibitor, Alfa Aesar, Harverhill, MA, United States). The tubes were wrapped in foil for protection from light and incubated at 37°C in an orbital shaker at 150 RPM. An absorbance measurement at an optical density of 400 and 600 nm were taken at three timepoints, 16, 24, and 32 h, and the pH was tested for each hormone treatment using pH tests strips.

To determine fungal burden, adult male and female BALB/c mice (6 of each per sex, 6–8 weeks old obtained from Jackson Laboratories) were infected via intratracheal injection with 1 × 10⁴ CFU of H99S. Mice were injected intraperitoneally with a 2.5:1 mix of ketamine:xylazine to anesthetize them (5–10 mg/kg) prior to surgery. For the procedure, mice were placed on their back, their neck area was cleaned with alcohol and a small incision was made over the thyroid. The skin was gently pulled aside, and 50 μL of H99S was injected directly into the trachea using a bent tuberculin needle. The incision was closed using VetBond and the mice were kept warm and observed closely until they regained consciousness. Mice were euthanized using carbon dioxide overdose at day 7 post-infection and the spleen, lungs and brain were removed and homogenized. Homogenates were diluted and plated on YPD plates for 2 days at 37°C and colonies were counted to determine fungal burden.

To determine survival, adult male and female BALB/c mice (10 of each per sex, 6–8 weeks old obtained from Jackson Laboratories) were infected via intratracheal injection with 1 × 10⁴ CFU of H99S, as above, and observed over the course of infection. Any mouse in a moribund state and/or distress was euthanized using carbon dioxide overdose to avoid unnecessary suffering. Mice were monitored daily for mortality and morbidity and deaths or dates euthanized were recorded.

H99S cells (1 × 10⁵ cells/mL) were added to Dulbecco’s modified Eagle media containing testosterone, estradiol, or ethanol and incubated at 37°C + 5% CO₂ for 18 h, as described in Charlier et al. (2005). After incubation, supernatants were collected, centrifuged to remove C. neoformans cells, and stored at −20°C until used to assess PA and GA concentrations by ELISA (see section “ELISA to Detect Pantothenic and Gibberellic Acid”).

Melanization studies in liquid L-DOPA media over 5 days and dose-response curves were analyzed using linear regression analysis. Conditioned media experiments and serial dilutions were analyzed using multivariate analysis of variance (MANOVA) with simple contrasts. ELISA data were analyzed using Tukey Kramer’s honest significant difference for multiple comparisons and MANOVA with simple contrasts. qRT-PCR data were analyzed using the 2^–ΔΔC′T method (Rao et al., 2013) for relative gene expression analysis. RNA sequencing data were analyzed using edgeR differential expression analysis using R studio (Boston, MA, United States). Fungal burden data and survival were analyzed using Wilcoxon Ranks Sums test. Statistical analysis was conducted using JMP, version 14 (SAS Institute, Cary, NC, United States). p < 0.05 was considered significant.

Initial data suggested that the melanization rate of C. neoformans could be influenced by steroid hormones. As shown in Figure 1A, a number of C. neoformans clinical isolates were grown on L-DOPA agar plates supplemented with steroid hormones for 7 days to test this hypothesis. Visually, there was a clear difference in melanization when C. neoformans was in the presence of testosterone. To determine if testosterone was perhaps a substrate for melanization, cells were grown on plates containing testosterone or estrogen without L-DOPA; the cells did not melanize in the presence of either hormone (data not shown). Thus, to derive quantitative data of this difference, the wild-type H99S C. neoformans strain was grown in liquid L-DOPA medium supplemented with steroid hormones or ethanol as a vehicle control for 5 days. Experiments to test for an effect of ethanol did not show any difference in fungal growth or melanization (data not shown). Each day the absorbance at an optical density of 400 and 600 nm (to correct for cell density) was measured. An increase in melanization when C. neoformans was in the presence of testosterone (p < 0.001) became apparent from day one and this trend continued throughout day five, except for day four, where there was no significant difference between any of the treatments (Figure 1B).

The regulation of gene expression in response to population density is a phenomenon known as quorum sensing (Nealson and Hastings, 1979). Because we hypothesized that a QSM might be responsible for the observed difference in melanization, to determine if C. neoformans’ known QSM, PA, affected melanization when steroid hormones were present, a conditioned media experiment was conducted. Conditioned media is the supernatant of cells grown in media for 7 days; thus any secreted QSM that increased growth rate would be found in this supernatant. If C. neoformans was producing a QSM that affected melanization, an increase in melanization when conditioned media was used would be expected. To test this hypothesis, the wild type H99S strain was grown in conditioned media for 32 h. The absorbance at an optical density of 400 and 600 nm was measured at three time points, 16, 24, and 32 h. The pH of each treatment was also tested using pH test strips and showed no difference in pH (data not shown). In the previous 5-day experiments, the variation in melanization occurred at day three; however, when conditioned media was used, this variation was seen at 16 h (Figures 1C,D). There was also a significant increase in melanization (p < 0.002) when C. neoformans was in the presence of testosterone compared to ethanol or estrogen (Figures 1C,D).

To further determine the effects of steroid hormones on QSM secretion and melanization, a dose-response analysis of both testosterone and estradiol was completed in conditioned media. Five concentrations of testosterone (0, 0.5, 2, 5, and 10 ng/mL) and estradiol (0, 100, 200, 400, and 10 ng/mL) were used to supplement conditioned L-DOPA media. These concentrations were chosen as a range of physiological concentrations. H99S was grown for 32 h in the various hormone concentrations and the absorbance at an optical density of 400 and 600 nm was measured (Supplementary Figures 1A,B). As the concentration of both testosterone and estradiol increased, melanization also increased in C. neoformans (p = 0.02), indicating that both hormones affected melanization in C. neoformans.

ELISAs were completed on H99S samples to determine the concentration of PA secreted in various conditions. When PA secretion was compared between C. neoformans in steroid hormone treatments over 5 days of melanization, there was no difference between testosterone and estradiol, and both hormones affected PA secretion when compared to the ethanol control (Figure 2A). To determine if the presence of hormones affected fungal growth, which might explain the increased secretion of PA, we conducted a growth curve of H99S in the presence of steroid hormones. While there was similar growth in the presence of testosterone and estrogen, there was significantly increased growth in both hormones compared to growth in ethanol (Supplementary Figure 2), suggesting that steroid hormones were likely partly responsible for the increased PA secretion. There was significantly more PA present at day five compared to previous days in all hormones, as expected, due to the cell density increase (p = 0.01). ELISAs completed on conditioned media (Figure 2B) and steroid hormone dose-response samples (Figure 2C) also indicated no difference in PA secretion when comparing testosterone to estradiol (except for one concentration in the dose-response curves), suggesting that PA secretion was not causing the melanization difference observed during the initial experiments.

Due to the known effects of steroid hormones on gene regulation (Parker, 1988; Ing, 2005), we hypothesized that testosterone’s effects were at the transcriptional level. Since it was initially thought that the observed melanization difference between testosterone and estrogen could be due to a QSM, qRT-PCR was utilized to determine gene expression of genes identified from the literature, which were known to be either involved in C. neoformans melanization or were C. neoformans homologs of other known QSMs (Hornby et al., 2001; Eisenman et al., 2007 (Table 1). The testosterone- and estrogen-treated samples were compared to the ethanol-treated samples (the vehicle control). When C. neoformans was in the presence of testosterone, these genes were upregulated compared to when C. neoformans was in the presence of estradiol. Thus, to determine changes in the entire transcriptome, RNA sequencing was pursued.

RNA sequencing analysis comparing C. neoformans in the presence of steroid hormones was used to identify gene expression changes across the transcriptome during melanization. To determine what time point was best to extract RNA, qRT-PCR was conducted on levels of laccase mRNA in cells grown in L-DOPA media for 1, 2, or 3 days. Laccase mRNA was upregulated on day 1, but not on days 2 or 3 (data not shown). However, since C. neoformans cells visually started to melanize on day 3 (Figure 1A), it was decided to extract RNA from C. neoformans on day three. Then, cDNA libraries were prepared and sent for sequencing. For data analysis, the testosterone-treated samples were compared to both the estrogen-treated and ethanol-treated samples. These data revealed four genes that showed significant upregulation in C. neoformans in the presence of testosterone compared to estrogen (Table 2). CNAG_03340, a flavonol synthase, is a transmembrane protein that is part of the gibberellic biosynthesis pathway (FungiDB, 2019). Given this finding, other genes in the gibberellic biosynthesis pathway in the RNA sequencing data were identified, including four additional genes: cytochrome p450 monooxygenase pc-2 (CNAG_02841), cytochrome p450 (CNAG_05842 and CNAG_04029), and sphingolipid delta-4 desaturase (CNAG_00644) (Table 2). The gibberellic biosynthesis pathway produces GA, which typically increases growth rates in other organisms (Hedden et al., 2001; Tudzynski, 2005). This pathway has not been previously identified in C. neoformans.

RNA sequencing analysis revealed multiple genes that were part of a gibberellic biosynthesis pathway that were upregulated during melanization of C. neoformans in the presence of testosterone. ELISAs were completed on multiple H99S samples to determine if GA was secreted by C. neoformans and at what concentration. Basal levels of GA secretion by C. neoformans were first determined in L-DOPA media without steroid hormones over a 3-day period. C. neoformans secreted similar levels of GA per day: 13 ng/mL on day 1, 15 ng/mL on day 2, and 16 ng/mL GA on day 3. These data were similar to those measured in the presence of ethanol during a 5-day melanization experiment (Figure 3A) and a conditioned media experiment (Figure 3B). ELISAs of these experiments showed a significant increase in GA secretion when C. neoformans was in the presence of testosterone compared to estradiol (p < 0.05). When comparing ELISAs completed on the dose-response of both testosterone and estradiol, the difference in GA secretion was striking (Figure 3C). When C. neoformans was in the presence of testosterone, there was a dose-dependent increase in GA secretion as testosterone concentration increased. These data were similar to the PA ELISA completed on the testosterone dose-response (Figure 2C). However, when C. neoformans was in the presence of estradiol there was no change in GA secretion when estradiol concentration varied. This led to the hypothesis that testosterone was regulating GA secretion, and this secretion could be causing the difference in melanization observed when hormones were present.

We further hypothesized that GA could be acting as a QSM, similar to PA. However, when this hypothesis was tested, it was found that GA was not a QSM. Figure 4A reveals ELISAs completed on C. neoformans at different cell densities in the presence of steroid hormones. As cell density increased, PA concentrations also increased (p < 0.001), as expected for a QSM. However, GA concentrations remained the same between cell densities (p > 0.05) (Figure 4B); revealing that GA was not a QSM, but instead could potentially function as a growth hormone whose production was regulated by testosterone; thus, affecting melanization.

To determine if GA was acting as a growth hormone and to understand the effects GA had on C. neoformans, exogenous GA was added to H99S samples without the presence of steroid hormones. Growth experiments showed that addition of GA significantly increased the optical density, suggesting GA was acting as a growth hormone (p < 0.0001 for all days, Figure 5A). Melanization experiments revealed that when C. neoformans was in the presence of exogenous GA, there was an increase in melanization compared to H99S alone (p < 0.0001, Figure 5B). A dose-response using various concentrations of GA was also completed (Figure 5C) showing that as GA concentration increased, melanization of C. neoformans also increased (p < 0.02). This indicated that GA had an effect on melanization, likely as a growth hormone, and when secreted, resulted in increased cell density.

The GA pathway in C. neoformans has yet to be discussed in the literature. However, this growth hormone is found in other fungi, such as Gibberella fujikuroi (McInnes et al., 1977), making it more likely to be found in C. neoformans as well. Supplementary Figure 3 shows the GA pathway in Gibberella fujikuroi and where flavonol synthase is located in the pathway. This gene was identified in the RNA sequencing data and was significantly upregulated when C. neoformans was in the presence of testosterone.

To determine if the GA biosynthesis pathway was involved in melanization, KO strains of genes identified in the RNA sequencing analysis, including various genes involved in the GA biosynthesis pathway, were tested for amounts of melanization in L-DOPA media plus testosterone (over 5 days) compared to H99S plus testosterone. All of the KO strains tested in liquid medium showed less melanization over 5 days (Figure 6A) when compared to H99S plus testosterone, and the KO strains of genes involved in the GA biosynthesis pathway showed the least amount of melanization. If C. neoformans was producing GA it was likely that these genes (cytochrome p450 monooxygenase pc-2, flavonol synthase, the hypothetical proteins, and cytochrome p450) all played a role in C. neoformans melanization in testosterone. A growth curve of the KO strains was also completed to compare growth rates in the KO strains to the H99S wildtype strain in L-DOPA media (Supplementary Figure 4). All of the KO strains grew significantly slower than the wildtype strain, H99S, regardless of the addition of exogenous hormone, which could be confounding the results. However, at the 5-day time point, the color of the KO strains were all a very light brown, indicating that these strains melanized, just at reduced levels (data not shown).

To determine the concentration of GA during melanization of the KO strains of genes in the GA biosynthesis pathway, ELISAs were conducted to determine the amount of GA secreted at 32 h in conditioned media (Figure 6B). GA concentrations varied widely when the KO strains were compared to H99S plus testosterone, so the concentrations were normalized by the optical density at 600 nm to control for cell density. In the KO strains, the normalized concentrations ranged from 10–20 ng/mL compared to 436 ng/mL in H99S plus testosterone. Additionally, the KO strains of the two hypothetical proteins that were identified in the RNA sequencing were also tested for GA secretion during melanization at 32 h (Figure 6C). As observed for the KO strains of genes in the GA biosynthesis pathway, these strains also showed reduced normalized secretion of GA compared to H99S plus testosterone (23–31 ng/mL compared to 436 ng/mL in H99S plus testosterone). These data suggested that both the hypothetical proteins and the genes in the GA biosynthesis pathway affected GA production.

To verify that the GA biosynthesis pathway was involved in melanization, another conditioned media experiment with exogenous inhibitors of GA and PA was conducted (Figures 7A–C). All treatments of H99S contained GA, chlormequat chloride (a GA inhibitor), PA, or DL-Serine (a PA inhibitor) plus the presence of steroid hormones. When exogenous GA and testosterone were added to C. neoformans, there was a significant increase in melanization compared to C. neoformans in the presence of exogenous PA and testosterone. Chlormequat chloride and DL-Serine are known to inhibit 50% of GA and PA production, respectively, in other organisms (Rademacher, 1992). This experiment demonstrated a significant decrease in melanization when chlormequat chloride or DL-Serine was present, suggesting that C. neoformans was producing both GA and PA molecules, and their production was being inhibited; thereby, decreasing melanization in C. neoformans.

We hypothesized that due to the GA concentration only being increased in the presence of testosterone compared to estradiol (Figure 3C) and the fact that GA had an effect on melanization (Figure 5B), testosterone was regulating GA production, which affected melanization, resulting in the melanization differences we observed when C. neoformans was in the presence of steroid hormones. RNA sequencing data revealed a hypothetical lncRNA (>200 nt) that was upregulated 88-fold when C. neoformans was in the presence of testosterone. When the lncRNA sequence was used to search for sequence homology using the BLAST algorithm (NCBI), multiple genes, including another gene identified in the RNAseq data (CNAG_05356), with transmembrane domains were identified (Table 3). CNAG_05356 is a hypothetical protein that shows decreased melanization and decreased GA secretion when knocked out, as shown in Figures 6A,C, indicating this gene is involved in GA secretion in C. neoformans.

Assuming testosterone enters C. neoformans cells, we hypothesize that testosterone upregulates transcription of GA and the hypothetical lncRNA is activated by testosterone to regulate transcription or translation of genes that encode proteins containing transmembrane domains that were identified in the BLAST search, and the RNA sequencing data, which then play a role in the transport and release of GA from the cell (Figure 8). Once GA is released from the cell, it acts as a growth hormone to increase proliferation of C. neoformans, which results in increased fungal growth and melanization, once a high enough cell density has been reached.

As GA production is required for the fungus G. fujikuroi to cause bakanae disease in rice (Studt et al., 2013), GA production may also contribute to pathogenesis in C. neoformans. To determine if GA was produced in other conditions than melanizing conditions, we incubated H99S in the presence of steroid hormones and 5% CO₂ to mimic the environment of the host lung and then measured the concentration of GA and PA in the supernatant the next day. There was significantly more GA secreted from C. neoformans incubated in the presence of testosterone (p < 0.0001) than any other condition, while there were significantly higher concentrations of PA secreted from C. neoformans incubated in the presence of estradiol and testosterone than in ethanol or media alone (p < 0.05, Figure 9A).

We next infected male and female BALB/c mice with H99S to determine if there was a difference in fungal burden in vivo in the presence of physiological levels of hormones. While there was no difference in survival or fungal burden in each individual organ (spleen, lungs, or brain) between males and females, at 7 days post-infection, males had a significantly higher overall fungal burden than females when the fungal burden from all tested organs was summed (p = 0.0345, Figure 9B).