The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i. e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation.
About 500 million years of evolution separate the fungal phyla Ascomycota and Basidiomycota [1,2]. The cell cycle is an essential biological process driving cell division of these distantly related yeasts, and therefore may be under strong selective pressure for conservation. Both Saccharomyces cerevisiae (Ascomycota) and Cryptococcus neoformans (Basidiomycota) can grow and divide asymmetrically in a budding yeast form. C. neoformans is a causative agent of deadly fungal meningitis, primarily in immune-compromised patients [3,4]. Many groups studying C. neoformans focus on virulence factors for human infection, such as the yeast’s polysaccharide capsule, melanin production, Titan cell formation, and others [5–9]. We propose that the function of cell-cycle regulators, which are essential for proliferation in the host, merit further investigation as virulence factors. Furthermore, there is evidence that virulence pathways are perturbed when cell-cycle progression is slowed, which suggests direct connections between cell-cycle regulators and virulence pathways [10,11]. The cell cycle is the process by which a cell duplicates its contents and faithfully divides into two genetically identical cells. In eukaryotes, a biochemical oscillator drives sequential cell-cycle events, where the cyclin-dependent kinase (CDK) and its variety of cyclin binding partners initiate events by phosphorylation, followed by destruction of kinase activity in mitosis by the anaphase-promoting complex (APC). Another common feature of the eukaryotic cell cycle is a temporally regulated program of transcription, which has been demonstrated in S. cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, mouse fibroblasts, and human tissue culture cells [12–22]. These programs of periodic genes include cyclin mRNAs, DNA replication factors, APC activators, and other cellular components that are utilized at specific times during the cell cycle. Our group and others have proposed that this “just-in-time transcription” mechanism is an important aspect of energy-efficient and faithful cell divisions [23,24]. In S. cerevisiae, an interconnected network of periodic transcription factors (TFs) is capable of driving the periodic program of cell-cycle gene expression [15,25–27]. Aspects of this yeast TF network are conserved in human cells; for example, G2/M genes are activated by a periodic forkhead domain-containing TF in both eukaryotes [22,28]. The topology of cell-cycle entry is also functionally conserved, where a repressor (S. c. WHI5, H. s. RB1) is removed by G1 cyclin/CDK phosphorylation to activate a G1/S transcription factor complex (S. c. SBF/MBF, H. s. E2F-TFDP1) [29]. However, the genes involved in cell-cycle entry are not conserved at the sequence level between fungi and mammals [30], suggesting that the fungal pathway could be targeted with drugs without affecting mammalian host cells. Sequence-specific DNA-binding TFs have been identified in C. neoformans and phenotypically profiled by single gene knockouts [6,31,32]. This TF deletion collection was profiled over many virulence factor-inducing conditions to discover pathways that regulate disease and drug response genes [32]. Serial activation of TFs during capsule production has also been studied to elucidate the order in which TFs control virulence gene products [31]. However, the cell cycle has not been investigated in synchronous populations of cells to date. Although the phenotypes of some single mutant cell-cycle TFs have been examined from asynchronous populations, these studies offer limited understanding of temporal aspects of gene expression during the cell cycle. Here we investigate transcriptional dynamics of the pathogenic yeast C. neoformans using cells synchronized in the cell cycle. We compare our findings to the cell-cycle transcriptional program in S. cerevisiae. We find that a similar percentage of all genes (~20%) are periodically transcribed during the cell cycle, and we present a comprehensive periodicity analysis for all expressed genes in both yeasts. We show that S-phase gene orthologs are highly conserved and temporally precede M-phase gene orthologs in both yeasts. Additionally, we find that many TFs in the cell-cycle entry pathway are conserved in sequence homology, periodicity, and timing of expression in C. neoformans, while others, notably genes involved in budding, are not. We also identify 40 virulence genes that appear to be cell-cycle-regulated, along with nearly 100 orthologous fungal genes that are periodic in the same cell-cycle phase. Taken together, these cell-cycle genes represent candidates for further study and for novel antifungal drug development.
Identifying approaches for synchronizing populations of C. neoformans has been challenging. We succeeded in synchronizing by centrifugal elutriation, a method that has been very successful for S. cerevisiae cells [15,27,33]. For C. neoformans, we isolated early G1 daughter cells by centrifugal elutriation and released the population into rich media (YEPD) at 30°C to monitor cell-cycle progression, as described previously [34]. This size-gradient synchrony procedure is conceptually similar to the C. neoformans synchrony procedure presented by Raclavsky and colleagues [35]. For S. cerevisiae, we isolated G1 cells by alpha-factor mating pheromone treatment [36]. We utilized this synchrony technique to isolate larger S. cerevisiae cells and to offset some loss of synchrony over time due to asymmetric cell divisions. A functional mating pheromone peptide for C. neoformans has been described but is difficult to synthesize in suitable quantities [37]. After release from synchronization, bud formation and population doubling were counted for at least 200 cells over time (Fig 1). The period of bud emergence was about 75 minutes in both budding yeasts grown in rich media, although the synchrony of bud emergence after the first bud in C. neoformans appeared to be less robust (Fig 1A and 1B). Each yeast population completed more than two population doublings over the course of the experiments. Total RNA was extracted from yeast cells at each time point (every 5 minutes for S. cerevisiae, or every 10 minutes for C. neoformans) and multiplexed for stranded RNA-Sequencing. Between 87–92% of reads mapped uniquely to the respective yeast genomes (S1 File). To identify periodic genes, we applied periodicity algorithms to the time series gene expression datasets. Four algorithms were used to determine periodicity rankings for all genes in each yeast: de Lichtenberg, JTK-CYCLE, Lomb-Scargle, and persistent homology [38–42]. Since each algorithm favors slightly different periodic curve shapes [43], we summed the periodicity rankings from each algorithm and ranked all yeast genes by cumulative scores for S. cerevisiae and for C. neoformans (S1 Table and S2 Table, respectively). By visual inspection, the top 1600 ranked genes in both yeasts appeared periodically transcribed during the cell cycle (S1 Fig). There was no clear “threshold” between periodic and non-periodic genes during the cell cycle—rather, we observed a distribution of gene expression shapes and signatures over time (S1 Fig). Previous work on the S. cerevisiae cell cycle has reported lists ranging from 400–1200 periodic genes. To validate our RNA-Sequencing time series dataset for the S. cerevisiae cell cycle, we compared the top-ranked 1600 periodic genes to previously published cell-cycle gene lists and found a 57–89% range of overlap with previous periodic gene lists (S2 Fig) [12–15,33,41,44,45]. Three filters were applied to each budding yeast dataset to estimate and compare the number of periodic genes (S1 File). First, we pruned noisy, low-expression genes from each dataset, leaving 5913 expressed genes in S. cerevisiae (S1 Table) and 6182 expressed genes in C. neoformans (S2 Table). Next, we took the top 1600 expressed genes from the cumulative ranking of the four periodicity algorithms described above. Finally, we applied a score cutoff to each list of top 1600 genes using the Lomb-Scargle algorithm (see S1 File) [39,40,43]. We estimated that there are 1246 periodic genes in S. cerevisiae (~21% expressed genes) and 1134 periodic genes in C. neoformans (~18% expressed genes) (Fig 2). We also provided multiple criteria for evaluating the cell-cycle expression patterns of individual genes in each yeast (S1 Table, S2 Table, S1 Fig). Cellular processes that contribute to virulence are a major focus of work in the C. neoformans field. We took advantage of the partial C. neoformans deletion collection and genetic screens for virulence factors [6] and searched for periodic virulence genes. We found that 40 genes (about 16% of the virulence genes characterized by the Madhani group and many previous studies) were periodically expressed in C. neoformans during the cell cycle (S3 Table). These virulence genes are periodic during normal cycles in rich media, which suggests that some virulence processes are directly cell-cycle-regulated. For example, budding and cell wall synthesis are coupled to cell-cycle progression in S. cerevisiae. A subset of 14 periodic virulence genes in C. neoformans had capsule and/or cell wall phenotypes reported in previous studies (S3 Table). We then asked if the 40 periodic virulence genes might be co-regulated during the C. neoformans cell cycle (S3 Fig). Over half of the periodic virulence genes clustered together and peaked in a similar cell-cycle phase (20–30 minutes into cycle 1). 11 of the 14 capsule / cell wall genes were contained in this cluster (S3 Fig, S3 Table). Next, we wanted to ask if periodicity and temporal ordering of orthologous genes is evolutionarily conserved between the two budding yeasts. We compiled the largest list to date of putative sequence orthologs between C. neoformans and S. cerevisiae from the literature, databases, and additional BLAST searches (S1 File, S4 Table) [32,46–48]. About half of the periodic genes from each yeast (Fig 2) had at least one sequence ortholog in the other species. However, there were only about 230 pairs of orthologous genes that were labeled periodic in both yeasts. Those pairs of periodic orthologs have diverged in temporal ordering between C. neoformans and S. cerevisiae (Fig 3, S5 Table). These results indicated that the programs of periodic gene expression, and possibly the regulatory pathway, have diverged to some degree between the two budding yeasts. This altered temporal ordering between S. cerevisiae and C. neoformans periodic orthologous genes was likely not due to the experimental synchrony procedure. We obtained transcriptome data from two previous studies on S. cerevisiae cell-cycle-regulated transcription (which applied a different cell-cycle synchrony procedure, used different lab strains of S. cerevisiae, and/or measured gene expression on different platforms), and our list of periodic S. cerevisiae genes maintained temporal ordering during the cell cycle in all three datasets (S4 Fig). Cell-cycle regulated gene expression has also been investigated in a species of pathogenic Ascomycota, Candida albicans [49]. To ask about common periodic gene expression in an evolutionarily intermediate budding yeast species, we further identified putative periodic orthologous genes shared between S. cerevisiae, C. neoformans, and C. albicans. A core set of almost 100 orthologs appeared to have both conserved periodicity and temporal ordering between all three budding yeasts (S5 Fig, S5 Table). This fungal gene set was enriched for functions in mitotic cell cycle and cell-cycle processes, which suggested that core cell-cycle regulators are under strong selection for conservation at the sequence level and by timing of periodic gene expression. We reasoned that some cell-cycle events must be invariable in temporal ordering between fungi (S5 Fig). DNA replication (S-phase) should be highly conserved across organisms because duplication of genetic material is essential for successful division. Segregation of genomic content during mitosis (M-phase) is also essential for division, and duplication must precede division. Using annotations for S. cerevisiae [50] we identified lists of genes known to be involved in regulating events in various cell-cycle phases including bud formation and growth [51,52], DNA replication [53,54], and spindle formation, mitosis, and mitotic exit [55–58]. We filtered the resulting gene lists by periodicity in S. cerevisiae (Fig 2A, S6 Table). We then identified orthologous genes in C. neoformans without enforcing a periodicity filter. We have previously shown that expression timing of canonical cell-cycle orthologs in S. cerevisiae and S. pombe can vary—some gene pairs shared expression patterns while others diverged [59]. To temporally align orthologous gene plots between S. cerevisiae and C. neoformans, we used the algorithmic approach described previously with S. cerevisiae and S. pombe time series transcriptome data [59]. The first, most synchronous cycle of budding data from each yeast was fit using the CLOCCS algorithm (Fig 1, S6 Fig) [59,60]. Time points in minutes were then transformed into cell-cycle lifeline points to visualize the data (see S1 File). As observed previously, S. cerevisiae genes that regulate budding, S-phase, and mitosis were largely transcribed periodically in the proper phases (Fig 4A, 4D and 4G) [12–15]. Cell-cycle gene expression peak time patterns were examined to quantitatively compare cell-cycle phases (S7 Fig). Bud assembly and growth genes peaked throughout the cell-cycle transcription program, and the temporal ordering of these genes repeated across cell cycles (Fig 4A, S7A and S7B Fig). Similarly, spindle assembly and mitosis genes peaked in the mid-to-late phases of the transcription program (Fig 4G). DNA replication genes peaked in a defined window in the middle phase of the transcription program (Fig 4D). We observed analogous expression patterns for C. neoformans orthologs associated with S-phase and mitosis (Fig 4E and 4H), but orthologs associated with budding appeared to be expressed with less restriction to a discrete cell-cycle phase or strict temporal order (S7 Fig). This budding gene pattern can be observed qualitatively where the unrestricted expression timing creates a more “speckled” appearance in the C. neoformans heatmap (Fig 4B) and differentially timed gene expression peaks (Fig 4C). We hypothesize that bud emergence and bud growth are not as tightly coordinated with cell-cycle progression in C. neoformans cells. Unlike S. cerevisiae where bud emergence occurs primarily at the G1/S transition, C. neoformans bud emergence can occur in a broad interval from G1 to G2 phases [61,62]. The difference in budding transcript behaviors between S. cerevisiae and C. neoformans orthologs could therefore reflect the difference in the cell biology of bud emergence and growth (Fig 4A and 4B). Only about 33% of the orthologous budding gene pairs were periodically expressed in C. neoformans, compared to 53% DNA replication and 61% mitosis orthologs (Fig 4B, 4E and 4H). Furthermore, budding orthologs that were periodic in both C. neoformans and S. cerevisiae showed some divergence in expression timing (Fig 4C). We also observed that bud emergence of C. neoformans cells during the time series appeared less synchronous in second and third cycles than S. cerevisiae cells (Fig 1A and 1B). Bud emergence in C. neoformans could be controlled by both stress pathways and TF inputs because the first budding cycle is highly synchronous after elutriation synchrony, which causes a transient stress response in released cells (Fig 1B). However, our data do not rule out a model where some budding genes in C. neoformans are controlled post-transcriptionally by localization, phosphorylation, or other periodic mechanisms. It is also possible that budding orthologs are more difficult to identify than other cell-cycle genes due to sequence divergence or that novel budding genes have evolved in the C. neoformans lineage. We have previously shown that a network of periodically expressed TFs is capable of driving the program of periodic genes during the S. cerevisiae cell cycle [15,27]. We hypothesized that a network of periodic TFs could also function in C. neoformans to drive a similar fraction of cell-cycle genes. Thus, the temporal re-ordering of part of the C. neoformans gene expression program (Fig 3) could be explained by two models: evolutionary re-wiring of shared network TFs with S. cerevisiae or novel TF network components arising in C. neoformans to drive cell-cycle genes. First, we asked if network TFs were conserved from S. cerevisiae to C. neoformans. Indeed, a majority of network TFs and key cell-cycle regulators have putative orthology between the two yeasts (Table 1) [30]. As observed for other cell-cycle genes (Fig 4), orthologs of some network TFs were expressed in the same phase in both yeasts, while others were expressed at different times (Table 1). Second, we asked if there were any novel periodic TFs in C. neoformans (i. e. TFs with no predicted ortholog in S. cerevisiae, or TFs with an ortholog in S. cerevisiae that is not known to function in the TF network). We constructed a list of periodic C. neoformans TFs by filtering a previously annotated transcription factor list [32] with our list of periodic genes (Fig 5, S7 Table). Indeed, 30 novel TF genes were periodic during the C. neoformans cell cycle (Fig 5A). Taken together, results from Table 1 and Fig 5 suggested that both network TF re-wiring and novel periodic TFs in C. neoformans could explain the differential ordering of periodic genes during the cell cycle (Fig 3). Putative S-phase regulators in C. neoformans exhibited transcript behaviors that were very similar in periodicity and in ordering to their S. cerevisiae orthologs (Fig 4D–4F). Thus, we predicted that the network motifs and TFs controlling the transcription of periodic S-phase genes could be conserved. Orthologous genes in the G1/S topology were largely conserved in periodic expression dynamics at cell-cycle entry (Fig 6). The expression timing of some genes had shifted earlier in the C. neoformans cell cycle (Fig 6C and 6F, Table 1), but this result does not refute the hypothesis that these genes are activated and functional at G1/S phase. Therefore, the network topology of cell-cycle entry appeared largely conserved in C. neoformans both by sequence and by gene expression dynamics. The prediction of this model is that a common G1/S transcriptional network drives a common set of S-phase periodic genes. To test this model, we examined promoter sequences from TF network genes in S. cerevisiae and C. neoformans, as well as the promoters of 38 periodic DNA replication ortholog pairs, and did an unbiased search for enriched TF binding sequences. The core motif “ACGCGT” for SBF/MBF transcription factors [63–65] was identified in both S. cerevisiae and C. neoformans promoters. The motif was not enriched in randomly selected periodic gene promoters, suggesting that SBF/MBF is functionally conserved in C. neoformans to drive TF network oscillations and DNA replication gene expression (S8 Fig).
Here, we present the first RNA-Sequencing dataset of transcription dynamics during the cell cycle of C. neoformans. Despite evolutionary distance between Basidiomycota and Ascomycota, S. cerevisiae and its extensive genome annotation provided an excellent analytical benchmark to compare to cell-cycle transcription in C. neoformans. RNA-Sequencing has been shown to be more quantitative than microarray technology for lowly- and highly-expressed genes using asynchronous S. cerevisiae cells due to microarray background fluorescence and saturation of fluorescence, respectively [66]. We demonstrate that 20% or more of all genes in the budding yeast genomes are periodically transcribed during the cell cycle. A ranking of periodicity for transcript dynamics in C. neoformans is provided (S2 Table). For the sake of comparison, we have presented gene sets of 1100–1200 periodic genes with the highest relative periodicity scores as “cell-cycle-regulated”; however, there is a continuum of periodic gene expression dynamics during the cell cycle in both yeasts (S1 Fig). The four periodicity algorithms applied here yielded a range of periodicity scores with no clear distinction between “periodic” and “non-periodic” gene sets (S1 and S2 Tables). These results suggest that yeast mRNAs fluctuate in expression with various degrees of cell-cycle periodicity. We propose that the top 20% periodic genes presented in this study are directly regulated by periodic cell-cycle TFs in C. neoformans and in S. cerevisiae. We also posit that some of the remaining 80% genes are weakly cell-cycle regulated. For example, some genes could be subject to complex regulation with one regulatory input from a cell-cycle periodic TF and another input from a constitutively expressed TF. We raise two important questions about the yeast periodic gene expression programs: is periodic expression of a core set (s) of genes required for the fungal cell cycle, and how are periodic gene dynamics controlled in each yeast? In both yeasts, periodic transcription is a high dimensional cell-cycle phenotype because transcriptional state reflects the phase-specific biology of the cell cycle over repeated cycles (Fig 2 and Fig 4). In other words, G1-, S-, and M-phase genes follow a defined temporal ordering pattern. S. cerevisiae cells synchronized by different methods and/or grown in different conditions display similar ordering of periodic cell-cycle genes, despite different cell-cycle period lengths (S4 Fig). Here, we examined the transcriptome of cycling C. neoformans cells at 30°C. Other groups have shown that C. neoformans cells spend more time in G1 phase at 24°C [67]. We predict that future studies examining cell-cycle transcription of C. neoformans cells grown in different conditions (i. e. non-rich media or 37°C infection temperature) would continue to display a similar temporal ordering of cell-cycle genes. These findings provide more evidence that “just-in-time transcription” is a conserved feature of eukaryotic cell cycles [23]. We show that some orthologous periodic genes have diverged in temporal ordering during the cell cycles of S. cerevisiae and C. neoformans over evolutionary time (Fig 3). We specifically investigated genes that play a role in bud emergence and bud growth, and we find that many budding gene orthologs are not controlled in a defined temporal order during the C. neoformans cell cycle (Figs 1A, 1B, 4A and 4B). On the other hand, DNA replication and mitosis genes do appear to be conserved by sequence homology, periodic expression, and temporal ordering (Fig 4D–4I). Lastly, we find that a set of about 100 orthologous genes is both periodic and expressed in proper cell-cycle phase in the budding yeasts S. cerevisiae, C. neoformans, and C. albicans (S5 Fig) [49]. These findings suggest that there may be a conserved set of fungal cell-cycle-control genes, which represent novel therapeutic targets for fungal infections. We posit that a network of periodic transcription factors (TFs) could control the periodic gene expression program in C. neoformans, which has been shown in S. cerevisiae and suggested in human cells [15,22,25,27]. Many orthologous genes to S. cerevisiae TF network components have diverged in expression timing in C. neoformans cells (Table 1). However, we show that the G1/S network topology is likely conserved between S. cerevisiae and C. neoformans because orthologous genes display similar expression dynamics (Fig 6). Furthermore, we find that the promoters of G1/S TF network orthologs and promoters of periodic DNA replication orthologs are enriched for an “ACGCGT” sequence motif, which matches the SBF/MBF binding site consensus in S. cerevisiae (S8 Fig) [63–65]. Therefore, we propose that the G1/S transcriptional motif—where a co-repressor is removed by G1 cyclin/CDK phosphorylation and a TF activator complex is de-repressed—is also conserved in C. neoformans (Fig 6B–6D and 6G) [29,30]. Downstream of the G1/S activator complex, the C. neoformans TF network may also contain a common forkhead domain S-phase activator and homeobox domain G1/S repressor (Fig 6E, Table 1) [14,68,69]. This partially conserved TF network model in C. neoformans explains the common G1/S topology, on-time DNA replication gene transcription, as well as differential expression of budding and other cell-cycle genes by divergent parts of the TF network. The regulation of periodic transcription and the function of a putative TF network warrant further investigation as virulence factors of fungal meningitis caused by C. neoformans. It has been previously shown that fluconazole drug treatment can affect cell ploidy in C. neoformans [70]. More recently, polyploid Titan cells were shown to produce haploid and aneuploid daughter cells during C. neoformans infection [71]. Therefore, future work on proper regulation of DNA replication and the contribution of periodic gene products could greatly benefit our understanding of genome stability in C. neoformans. The C. neoformans TF deletion collection was recently phenotyped, and the potential of targeted TF therapies was discussed [32,72]. We have added to the C. neoformans genotype/phenotype map by documenting the functional outputs of cell-cycle TFs over synchronized cell cycles. We also propose that a conserved G1/S topology of cell-cycle TFs may initiate the cell-cycle transcription network in C. neoformans. It is possible that a multi-drug combination targeting cell-cycle regulators and previously characterized virulence pathways could yield more successful antifungal therapies [72]. For example, a combination therapy could target TFs at the conserved G1/S topology to slow cell-cycle entry and also target fungal cell wall or capsule growth. In the circadian rhythm field, it has been shown that drugs targeting Clock Controlled Genes are most potent when administered at the time of the target gene’s peak expression [73]. Interestingly, deletion of the known SBF/MBF ortholog, Mbs1 (CNAG_07464), is viable in C. neoformans [32,74]. These genetic results do not match S. cerevisiae, where swi4 mbp1 double mutants are inviable [75]. In fact, deletion of the single known G1 cyclin ortholog, CNAG_06092, is also viable in C. neoformans [10]. Mbs1 and the G1 cyclin are likely important for cell-cycle progression in C. neoformans because mutant phenotypes are highly defective in capsule formation in G1 phase, melanin production, and response to Hydroxyurea treatment during S phase [10,11,32,74]. However, the genetics are inconsistent with findings in S. cerevisiae and warrant further investigation to characterize the G1/S TF network topology of C. neoformans. It is possible that uncharacterized, redundant genes exist in the C. neoformans G1/S network motif. We find that 40 candidate virulence genes are periodically expressed during the C. neoformans cell cycle (S3 Table, S3 Fig). An important direction for future work is to identify the mechanistic links between cell-cycle regulators and virulence pathways. 14 periodic virulence genes have annotated phenotypes in capsule formation and/or cell wall secretion. Fungal cells must secrete new cell wall and capsule during growth, and the direct links between cell cycle and these virulence factors in C. neoformans warrants further study because the cell wall and capsule are not present in host cells. The ultimate goal of this work is to identify the regulatory mechanism of periodic gene expression in C. neoformans and to find optimal drug targets and combination therapies for disrupting the fungal cell cycle.
The wild-type Saccharomyces cerevisiae strain is a derivative of BF264-15D MATa bar1 [76,77]. The wild-type Cryptococcus neoformans var. grubii serotype A strain is a derivative of H99F [47]. Yeast cultures were grown in standard YEP medium (1% yeast extract, 2% peptone, 0. 012% adenine, 0. 006% uracil supplemented with 2% dextrose sugar). For centrifugal elutriation, cultures were grown in YEP-dextrose (YEPD) medium at 30°C overnight. Elutriated early G1 cells were then resuspended in fresh YEPD medium at 30°C for time series experiments. For α-factor arrest, cultures were grown in YEPD medium at 30°C and incubated with 30 ng/ml α-factor for about 110 minutes. Synchronized cultures were then resuspended in fresh YEPD medium at 30°C. Aliquots were taken at each time point and subsequently assayed by RNA-Sequencing. Total RNA was isolated by acid phenol extraction as described previously [34]. Samples were submitted to the Duke Sequencing Facility (https: //www. genome. duke. edu/cores-and-services/sequencing-and-genomic-technologies) for stranded library preparation and sequencing. mRNA was amplified and barcoded (Illumina TruSeq Stranded mRNA Library Preparation Kit for S. cerevisiae and KAPA Stranded mRNA-Seq Library Preparation Kit for C. neoformans) and reads were sequenced in accordance with standard Illumina HiSeq protocols. For S. cerevisiae, libraries of 50 base-pair single-end reads were prepared, and 10 samples were multiplexed and sequenced together in each single lane. For C. neoformans, libraries of 125 base-pair paired-end reads were prepared (due to larger and more complex yeast transcriptome with introns), and 12 samples were multiplexed and sequenced together in each single lane. Raw FASTQ files were aligned to the respective yeast genomes using STAR [78]. Aligned reads were assembled into transcripts, quantified, and normalized using Cufflinks2 [79]. Samples from each yeast time series were normalized together using the CuffNorm feature. The normalized output FPKM gene expression levels were used in the analyses presented. A detailed description of each analysis pipeline is presented in the S1 File. RNA-Sequencing gene expression data from this manuscript have been submitted to the NCBI Gene Expression Omnibus (GEO; https: //www. ncbi. nlm. nih. gov/geo/) under accession number GSE80474.
The opportunistic fungal pathogen Cryptococcus neoformans infects immune-compromised humans and causes fungal meningitis by proliferating in the central nervous system. The cell cycle has not been studied at the whole transcriptome level in C. neoformans. Here, we present the expression dynamics of all genes from a synchronous population of C. neoformans cells over multiple cell cycles. Our study shows that almost 20% of all C. neoformans genes are periodically expressed during the cell cycle. We also compare the program of cell-cycle-regulated transcription in C. neoformans to the well-studied but evolutionary distant yeast, Saccharomyces cerevisiae. We find that many orthologous cell-cycle genes are highly conserved in expression pattern (e. g. DNA replication and mitosis genes), while others, notably budding genes, have diverged in expression ordering. We also identify 40 virulence genes from previous studies that are periodically expressed during the C. neoformans cell cycle in rich media. Our findings indicate that a conserved set of fungal transcription factors (TFs) controls the expression of conserved cell-cycle genes, while other periodic transcripts are likely controlled by species-specific TFs.
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Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans