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. Author manuscript; available in PMC: 2026 Jan 20.
Published in final edited form as: Nat Microbiol. 2018 May 21;3(6):698–707. doi: 10.1038/s41564-018-0160-4

Cryptococcus neoformans sexual reproduction is controlled by a quorum sensing peptide

Xiuyun Tian 1,†, Guang-Jun He 1,†, Pengjie Hu 1,2,†, Lei Chen 1,2, Changyu Tao 3, Ying-Lu Cui 4, Lan Shen 1, Weixin Ke 1,2, Haijiao Xu 1, Youbao Zhao 5, Qijiang Xu 6, Fengyan Bai 1, Bian Wu 4, Ence Yang 3, Xiaorong Lin 5, Linqi Wang 1,2,*
PMCID: PMC12813696  NIHMSID: NIHMS2137067  PMID: 29784977

Abstract

Bacterial quorum sensing (QS) is a well-characterised communication system that governs a large variety of collective behaviours. By comparison, QS regulation in eukaryotic microbes remains poorly understood, especially its functional role in eukaryote-specific behaviours, such as sexual reproduction. Cryptococcus neoformans is a prevalent fungal pathogen that has two defined sexual cycles (bisexual and unisexual) and is a model organism for studying sexual reproduction in fungi. Here, we show that the QS peptide Qsp1 serves as an important signalling molecule for both forms of sexual reproduction. Qsp1 orchestrates various differentiation and molecular processes, including meiosis, the hallmark of sexual reproduction. It activates bisexual mating, at least partially through the control of pheromone, a signal necessary for bisexual activation. Notably, Qsp1 also plays a major role in the intercellular regulation of unisexual initiation and coordination, in which pheromone is not strictly required. Through a multi-layered genetic screening approach, we identified the atypical zinc finger regulator Cqs2 as an important component of the Qsp1 signalling cascade during both bisexual and unisexual reproduction. The absence of Cqs2 eliminates the Qsp1-stimulated mating response. Together, these findings extend the range of behaviours governed by QS to sexual development and meiosis.

Sexual reproduction is a universal feature in eukaryotes, including fungi1, 2, 3. One of the model organisms used to study sexual reproduction is the prevalent human fungal pathogen Cryptococcus neoformans. C. neoformans has two sexual reproduction forms: bisexual reproduction taking place between cells of two compatible mating types (α and a) and unisexual reproduction (also termed haploid fruiting) involving only cells of the same mating type (mostly α)4. Like C. neoformans, its sibling pathogen Cryptococcus gattii has also been revealed to be able to undergo both bisexual and unisexual mating5. Previous observations have indicated a global dominance by α isolates in both C. neoformans and C. gattii6. This severe bias towards the α mating type probably leads to an infrequence of bisex in nature and mirrors the significance of α unisex in these pathogens. Developmentally, bisex and unisex undergo similar sequential differentiation processes, including filamentation, basidial formation, meiosis, and sporulation7. However, these two sexual cycles can be differentiated by certain cellular events or morphological features. For instance, cell-cell fusion (syngamy) is essential for the generation of dikaryotic hyphae during bisexual development, and these filaments have special fused clamp cells7. By comparison, filaments are monokaryotic and clamp cells are unfused during unisexual reproduction, in which endo-replication or cell fusion-independent karyogamy events but not syngamy is presumed the major approaches for ploidy change7, 8.

The mating MAPK cascade is the major signalling pathway for both bisex and unisex9, 10, and many components of this pathway, such as HMG regulator Mat2, are required for both reproduction modes. Their disruption not only abolishes or substantially impairs a-α cell fusion during bisex but also prevents monokaryotic filamentation (also termed self-filamentation) during haploid fruiting11, 12 . In contrast, some upstream components critical for the paracrine induction of bisex (Fig. 1a), including α pheromone Mfα, G-protein-coupled receptor Ste3α, and pheromone transporter Ste6, appear to be not strictly required for unisexual mating. The corresponding mutants in the JEC21α background (mf1,2,3αΔ, ste3αΔ and ste6Δ) have been found to be severely defective in bisexual mating but to retain partial or full capability to undergo self-filamentation13, 14, 15, 16. This led us to ask whether other paracrine systems contribute to the activation and coordination of the Cryptococcus unisexual cycle.

Figure 1 |. The mating pheromone is largely dispensable for unisexual reproduction in the XL280α background.

Figure 1 |

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a, Schematic diagram depicting proposed pheromone auto/paracrine regulation during bisexual and unisexual mating, respectively. During bisexual mating, the pheromone Mfa is produced by a cells, in which the transporter Ste6 facilitates its secretion. Secreted pheromone strongly induces mating response by binding to the compatible GPCR receptor Ste3α of α cells9, 13. The compatibility of Mfα and Ste3α during α unisexual reproduction remains unclear. b, The effect of the absence of various components of mating MAPK signalling and the CQS1 gene on aerial hyphal morphogenesis at the colony level during unisexual development. MAPK: the mitogen-activated protein kinase (Cpk1); MAPKK: MAPK kinase (Ste7); MAPKKK: MAPK kinase kinase (Ste11α); MAPKKKK: MAPK kinase kinase kinase (Ste20α). The hyphae in different strains were photographed after 10 days of growth. Scale bars: 1 mm. c, The effect of the absence of components of pheromone signalling and the CQS1 gene on sporulation during unisexual development. The spore chains in different strains were photographed after one week of growth. Scale bar: 20 μm. d, Pheromone is not required for expression of Cfl1 during unisexual reproduction based on images (Left) and quantification of fluorescence intensity (Right). Graph shows mean fluorescence intensity of two colonies for each strain. Scale bars: 1 mm. e, Diagram shows quantitative analysis method for paracrine induction of unisex based on the confrontation assay (Left). Recipient strain was dropped in proximity to pre-incubated donor colony and incidence of forming filamentous mini-colonies in recipient strain was recorded. The mini-colonies in different recipient strains were photographed 16 hrs after inoculation of the recipient (Right). Scale bars: 100 μm. f, Cells of XL280α and the cqs1Δ mutant were respectively plotted on mating-inducing medium (V8 agar) at different concentrations. FF was calculated based on the percentage of filamentous mini-colonies 21 hrs after mating induction. n = 3 independent experiments, mean ± SEM. In b,c,e, images are representative of more than five independent experiments conducted with similar results.

Results

The mating pheromone Mfα is largely dispensable for unisexual initiation and coordination in XL280α.

We first deleted all three pheromone genes and generated the pheromoneless mutant (mf1,2,3αΔ) in XL280α, which was chosen in the study because of its well-described ability to undergo robust unisexual development17. This aspect enabled us to sensitively assess various phases during unisexual cycle in this mutant. We showed that the absence of the mating pheromone in XL280α did not result in evident impairment in the sequential differentiation events associated with unisexual development, including self-filamentation and sporulation (Fig. 1b–c). Consistently, the absence of the pheromone Mfα only slightly affected the expression of the extracellular matrix signal Cfl1, a filament-specific molecular indicator18,19, which was nearly undetected in the mat2Δ mutant (Fig. 1d). In contrast, self-filamentation in XL280α was not observed in the mutants disrupting the gene encoding the downstream MAPK cascades (Fig. 1b). This echoes the phenotypes observed in the JEC21α-derived counterpart mutants11.

Quorum sensing peptides induce unisex.

We next sought to examine whether there is another intercellular communication system that could induce unisex in C. neoformans. For this, we used a quantitative approach based on confrontation analysis to evaluate the stimulatory effect by the potential unisex-inducing signal (Fig. 1e). We found that neither the absence of Mfα in the donor nor mutating STE3α in the recipient can attenuate the intercellular induction of filamentation (Fig. 1e). This result suggested that an unknown factor, rather than the α pheromone, intercellularly induces unisexual mating. Intriguingly, the production of this unknown signal correlated with cell density, and the filamentous mini-colony population achieved ~100% when the cells grew at a high cell density on mating-inducing medium (Fig. 1f). This prompted us to test whether the unknown factor inducing the unisex is a quorum sensing (QS) molecule due to its common role in sensing population density20. We thus assessed the previously reported Cryptococcus QS signals (pantothenic acid and the quorum sensing peptides, including Qsp1, Qsp2, and Qsp3)21, 22 for their activities to stimulate self-filamentation. Among these signals, only Qsp1 and Qsp2 exerted striking induction activity (Fig. 2a). A previous study has indicated that Qsp1 and Qsp2 can rescue the cell density-associated growth defect in a serotype D tup1Δ strain21, which fails to grow at low cell density. A more recent investigation has shown that Qsp1 plays an important role in mediating Cryptococcus virulence23. However, it remains unknown about the functional role of the QS peptides in sexual reproduction, which is considered to play an important role in promoting Cryptococcus infections2,4.

Figure 2 |. Quorum sensing peptides coding gene CQS1 is important for both bisexual and unisexual development.

Figure 2 |

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a, Effect of previously reported Cryptococcus QS signals on activation of filamentation during unisexual reproduction. 50 μM signals were used in the test. PA: pantothenic acid. Data shown is from two independent experiments. b, Paracrine induction of unisexual initiation needs the peptide signals encoded by CQS1. Scale bar: 100 μm. Representative images of n = 5 experiments. c, CQS1 is important for expression of filamentation-specific marker protein Cfl1. Scale bars: 1 mm. Images are representative of three independent experiments conducted with similar results. d, Deletion of CQS1 resulted in the defective expression of Dmc1-mCherry in basidia during unisexual mating which can be restored by the supplement of synthetic Qsp1 (final concentration: 50 μM) into the medium. The images of the fluorescence labeled strains were taken at 7 days post mating stimulation. For each strain, 50 basidia were examined for the expression of Dmc1-mCherry. ND: undetected. Errors bars indicate mean ± SD. Scale bars: 10 μm. e, Disruption of CQS1 blocked sporulation during unisexual mating. The cqs1Δ mutant sporulated in the presence of synthesized Qsp1. Scale bar: 20 μm. f, Cross between XL280α and XL280a led to profuse filamentation and robust sporulation. Deletion of CQS1 substantially attenuated filamentation and abolished sporulation during bisex. These defects were restored by the addition of synthesized Qsp1. Scale bars: 1 mm (upper panel), 20 μm (bottom panel). g, The addition of Qsp1 restored the defective expression of Dmc1-mCherry in cqs1 × cqs1 mutant cross. The fluorescence-labeled isogenic α and a strains were pre-mixed and the mixtures was subsequently plotted on V8 medium to stimulate mating. The images were taken at 7 days post mating stimulation. Scale bar: 10 μm. In e-g, images are representative of more than five independent experiments conducted with similar results.

CQS1 is important for activation and coordination of both bisexual and unisexual development.

Peptides Qsp1, Qsp2 and Qsp3 are different isoforms, maturated from three alternatively spliced transcripts from the gene, CQS121 (Supplementary Fig. 1a). Disruption of CQS1 substantially inhibited initiation of unisexual filaments (Fig. 1f and 2b). After extended incubation, only sparse unisexual filaments (mostly invasive hyphae) were observed in the cqs1Δ mutant (Fig. 1b). Furthermore, expression of the filamentation marker Cfl1 was greatly attenuated in the cqs1Δ mutant (Fig. 2c). The defects of filamentation and Cfl1 expression in the cqs1Δ mutant was rescued by synthesized Qsp1 and Qsp2, but not by Qsp3 (Supplementary Fig. 1b–c). This suggests that QS peptides represent the important paracrine signals for unisexual activation. Consistently, confrontation assays indicated that the cqs1Δ mutant failed to efficiently stimulate self-filamentation in the adjacent wild-type XL280α strain (Fig. 2b). In detailed phenotypic analysis, we showed that deletion of CQS1 had little or no effect on the formation of unfused clamp cells and monokaryotic growth (the hallmarks of unisexual hyphae) during self-filamentation, but resulted in remarkably shorter filaments compared with the wild-type strain (Supplementary Fig. 2a–c). These data suggest that CQS1 affects both hyphal initiation and extension during unisexual development.

To examine whether QS peptide as the important signalling molecule controls unisexual meiotic reproduction or is merely involved in hyphal differentiation as a morphogen, we investigated its functional role in meiotic cycle, which is the hallmark of sex. Early studies have identified meiosis-specific recombinase Dmc1, whose expression is essential for meiotic progression and the formation of four meiotic spore chains in C. neoformans24, 25. Deletion of CQS1 resulted in the undetectable expression of mCherry-fused Dmc1 in basidia and completely blocked meiotic sporulation, even after prolonged incubation (Fig. 2d–e). Again, these defects can be compensated by synthesized Qsp1 (Fig. 2d–e).

To test whether CQS1-activated unisex is unique to the XL280α background, CQS1 was mutated in the JEC21α background, which can undergo haploid fruiting but less abundantly than XL280α17. The JEC21α-derived cqs1Δ mutant failed to filament, even after one-month incubation (Supplementary Fig. 3). Likewise, such failure was suppressed by synthesized Qsp1. Furthermore, we performed a bilateral mating assay to determine whether CQS1 is also important for bisexual reproduction. We showed that bisexual filamentation was substantially decreased in bilateral mating (α cqs1Δ × a cqs1Δ), in which neither sporulation nor the expression of Dmc1-mCherry was observed (Fig. 2f–g). As expected, these bisexual defects were restored by the synthesized Qsp1 (Fig. 2f–g). We further investigated the regulatory relationship between Qsp1 and the α mating pheromone in two sexual forms using qRT-PCR analysis. We found that deletion of CQS1 reduced the mRNA levels of Mfα-coding genes during both bisexual and unisexual mating, but not vice versa (Supplementary Fig. 4). Considering the critical role of Mfα in bisexual activation15, Qsp1 activates bisexual mating at least partially via its control of the α pheromone.

Qsp1 stimulates mating response through the control of Mat2.

To reveal the Cryptococcus genes controlled by CQS1, we performed transcriptomic analysis via high-coverage RNA-sequencing (RNA-seq). 109 genes were found to be differentially expressed in cqs1Δ compared with the wild-type strain during unisexual development (Fig. 3a and Supplementary Table 1). Among these, 103 genes were also differentially expressed in the cqs1Δ mutant grown in the presence of synthetic Qsp1 or Qsp2 compared with the cqs1Δ mutant grown in the absence of the synthetic peptides (Fig. 3a and Supplementary Table 1). Importantly, the regulons of Qsp1 and Qsp2, but not Qsp3, were enriched with genes involved in different processes associated with mating (Fig. 3b–c). This echoes the aforementioned observation that Qsp3 failed to stimulate unisex in the cqs1Δ mutant (Supplementary Fig. 1b). On the basis of RNA-seq data, we identified only the transcript isoform encoding the Qsp1 precursor and no other CQS1 transcript isoform for other peptides (Supplementary Fig. 5). This indicates the exclusive role of Qsp1 among CQS1-derived QS peptides during unisexual reproduction.

Figure 3 |. Qsp1 mediates mating response through the control of Mat2.

Figure 3 |

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a, Venn diagram analysis indicates that CQS1 regulates gene expression largely through the paracrine control exerted by its peptide product. The strains were cultured on V8 (pH = 7) agar with or without synthetic Qsp1 peptide (512 nM). b, Identification of GO terms associated with fungal mating among DEGs of the cqs1Δ mutant in the presence of different QS peptides (512 nM each). Gene ontology analysis was performed using the DAVID gene ontology program. c, Gene expression of C. neoformans cells under different conditions. Color bar represents log2 relative expression values versus column 1. d, CQS1 controls many mating-responsive genes. e, Transcriptional profiling of the cqs1Δ mutant responding to different amounts of synthetic Qsp1 (4, 16 and 512 nM). Color bar indicates log2 fold change values. Column “0” represents the cqs1Δ mutant without peptide treatment. f, Expression patterns of genes belonging to group I based on their differential expression pattern in response to Qsp1 with different amounts (4, 16 and 512 nM) and Qsp3 (512 nM) in the cqs1Δ mutant. Top and bottom panels indicate major expression pattern of genes in group I and the expression pattern of mating MAPK pathway genes, respectively. The average expression level across all genes with the major pattern is shown with red line. g, Overexpression of Mat2 and Znf2 can rescue the defect of filamentation in the cqs1Δ mutant. Left panel: gene expression of group I regulators under different conditions (n = 2 independent RNA-seq experiments). Right panel: filamentation frequency when group I regulators were respectively overexpressed in cqs1Δ mutant (n = 4 independent experiments). ‘X’ indicates regulatory gene controlled by Qsp1. h, Qsp1 positively regulates the expression of MAT2. Bars show mean ± SEM of four independent experiments. i, Comparison of CQS1-regulated and MAT2-controlled genes under mating inducing condition. Original transcriptome data of mat2Δ were obtained from a previous study12. In a-f and i, all RNA-seq data come from two independent experiments.

Among Qsp1 regulated genes, ~64% exhibited differential expression after transferring cells cultured under mating-repressing condition onto mating-inducing medium, and are referred to as mating-responsive genes. This percentage was much higher than the ratio of mating-responsive genes in the Cryptococcus genome (Fig. 3d). To further define the genes responsible for Qsp1-mediated mating response, the expression profiles of the cqs1Δ mutant responding to synthetic Qsp1 in different amounts (4, 16 and 512 nM) were compared, and three sets of genes were grouped based on the sensitivity of their transcriptional response to Qsp1, as revealed by RNA-seq analysis (Fig. 3e). Among these gene sets, we concentrated on the group of genes (group I) that showed transcriptional response to 4 nM Qsp1 in the cqs1Δ mutant. This concentration was chosen because it successfully induced unisexual filamentation in the cqs1Δ mutant at a level comparable to the wild-type strain (Supplementary Fig. 6). We systematically assessed the expression patterns of the genes belonging to group I according to their differential expression in response to different amounts of Qsp1 or Qsp3 (as a negative control) in the cqs1Δ mutant. The major expression pattern, which covered ~29% of the group I genes (Fig. 3f, top panel), greatly resembled that of the mating MAPK pathway members (Fig. 3f, bottom panel). Thus, the genes belonging to this group likely contain the major Qsp1 regulon members responsible for the induction of the mating response. Due to the generally crucial role of DNA-binding transcriptional factors (TFs) in mastering biological processes, we specifically focused on the six TFs present in group I (Fig. 3g and Supplementary Fig. 7a–c). These regulators were individually overexpressed in the cqs1Δ mutant and the resultant strains were tested for the capability to induce unisexual filamentation. We found that overexpression of two previously known regulators, Mat2 and Znf2, restored the defect of self-filamentation in the absence of CQS1 (Fig. 3g). Znf2 is a known master regulator of filamentation that functions downstream of Mat2 during unisexual mating12, 26. Therefore, Qsp1 probably upregulates Mat2, which activates Znf2 to execute morphogenesis. Indeed, we found that Qsp1 up-regulated the expression of MAT2 during unisexual mating (Fig. 3h) and there was a marked overlap between Qsp1 and Mat2 targets (Fig. 3i).

Many QS factors guide microbial behaviours by sensing external stimulation27. The transcriptomic data indicated that Qsp1 influenced the expression of a set of genes potentially responsible for glucose response (Fig. 3c). In some fungi, glucose as a key metabolic/nutritional signal modulates sexual development28. This promoted us to test whether glucose affects sexual differentiation and mating response in C. neoformans. We found that glucose strongly inhibited self-filamentation and invasive growth, a cell-substrate adhesion process coupled with filamentous differentiation in C. neoformans26 (Fig. 4a–c). In contrast, these morphological behaviours were readily observed in the presence of alternative carbon sources, such as galactose, but were completely eliminated in cells where CQS1 is absent (Fig. 4a–c).

Figure 4 |. Qsp1 participates in glucose starvation-induced mating response and morphogenesis.

Figure 4 |

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a, b, Carbon source-dependent filamentation (a) and (b) invasive growth. Strains were grown on YP agar containing different carbon sources for three days (representative images of n = 5 experiments). For invasive growth, the plates were washed with cold water, and the remaining invasive cells embedded in agar were photographed. Scale bars: 100 μm (a), 1 mm (b). c, Phenotype scores are represented in distinct colors based on semi-quantitative analysis targeting the phenotypes related to different carbon sources. Results represent at least three independent experiments. d, Fold induction of MAPK pathway genes during the incubation on the media containing different carbon sources in the presence or absence of Qsp1. Cryptococcus cells cultured under extremely mating-repressing condition (YPD liquid condition) were transferred onto solid plates containing glucose or non-favored sugar galactose as the sole carbon source for inducing mating response. Data shown is from two independent experiments.

We further used RNA-seq analysis to evaluate the effect of different carbon sources on the induction of mating MAPK genes in the presence or absence of Qsp1 (Fig. 4d and Supplementary Table 2). We found that multiple components of the mating MAPK signalling cascade were highly induced at 12 hours after incubation of XL280α cells on the plate containing galactose as the sole carbon source, but the induction was greatly attenuated in the absence of Qsp1. In contrast, when using glucose as the carbon source, no evident induction in the expression of mating MAPK pathway members was observed, regardless of the presence of Qsp1. Together, these results suggest that starvation for glucose stimulates the mating response in a Qsp1-dependent manner.

Atypical zinc-finger regulator Cqs2 is an important component of Qsp1 signalling during both bisexual and unisexual reproduction.

To identify the important components of the Qsp1 signalling pathway, we developed a multi-layered genetic screening method based on random insertional mutagenesis via Agrobacterium-mediated transformation (Fig. 5a). We generated ~42,000 insertional mutants in the XL280α parental strain and screened for a combination of phenotypes, including the defect in self-filamentation and an inability to respond to synthetic Qsp1. The genomic DNA of mutants meeting all criteria were extracted, pooled and subjected to AIM-seq (Agrobacterium-mediated Insertional Mutagenesis Sequencing) to identify the insertion sites29. Multiple independent genetic loci were identified (Supplementary Table 3), including OPT1, which encodes an oligopeptide transporter23. Most recently, Homer CM et al. has shown that in the serotype A cryptococcal strain H99, Opt1 as a critical member of Qsp1 cascade is responsible for the internalization of secreted Qsp123. We found that the phenotypes associated with the opt1Δ mutant in XL280α bore a striking resemblance to those observed in the cqs1Δ mutant, including the defect of unisexual activation and Cfl1 expression (Supplementary Fig. 8a–b). However, these defects cannot be restored by synthesized Qsp1, suggesting that the internalization of Qsp1 by Opt1 is likely a central mechanism essential for different processes mediated by Qsp1. Moreover, RNA-seq analysis revealed a great overlap between Opt1-regulated genes and Qsp1 targets (Supplementary Fig. 8c and Supplementary Table 4). The identification of OPT1 also points to the efficacy and specificity for our screening strategy. Besides, we explored an insertion in proximity to the start site of CNF00370 (designated as CQS2) transcript using AIM-seq in combination with high-coverage RNA-seq analysis (Fig. 5b). We further confirmed that CQS2 serves as an important component of Qsp1 signalling pathway during both bisex and unisex, on the basis of the following findings: i) its mRNA level was up-regulated by Qsp1 (Fig. 5b); ii) CQS2 deletion greatly impaired bisexual and unisexual filamentation, considerably decreased expression of Cfl1 during self-filamentation, and abolished meiotic sporulation during both bisex and unisex (Fig. 5c–f); iii) synthetic Qsp1 cannot complement these defects in the cqs2Δ mutant (Fig. 5c–f); iv) transcriptomically, ~42.9% targets of Cqs2 were also regulated by Qsp1 (Fig. 5g and Supplementary Table 5) and none of the targets of Cqs2 showed significant transcriptional response to Qsp1 in the cqs2Δ mutant during unisex (Fig. 5h). Together, these data suggest that Cqs2 is an important component of Qsp1 signalling pathway that promotes bisex and unisex.

Figure 5 |. Cqs2 as an important Qsp1 signalling cascade member promotes bisexual and unisexual mating.

Figure 5 |

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a, A schematic diagram depicting the multi-layered genetic screening method developed to identify Qsp1 signalling genes. Scale bars: 100 μm. b, Diagram of the CQS2 (CNF00370) locus in the XL280α background based on RNA sequence reads. The arrow indicates the location of the insertion site in the CQS2Tn mutant. TSP indicates the transcriptional start point of CQS2. Enriched RNA-seq signals visualized by Integrated Genome Browser (IGB) are representative of two independent experiments. c, Rescue effect of synthetic Qsp1 on the defects of unisexual filamentation in cqs2Δ mutant. Scale bar: 100 μm. d, Bisexual filamentation for a wild-type cross between XL280α and XL280a and cqs2Δ bilateral mutant crosses. All mating patches were spotted on V8 medium with or without synthetic Qsp1 (final concentration: 50μM). Scale bar: 1 mm. e, CQS2 is essential for bisexual and unisexual sporulation. Bisexual sporulation was assessed in wild-type (XL280α × XL280a), and bilateral (α cqs2Δ × a cqs2Δ) mating assays on V8 medium with or without synthetic Qsp1 for 7 days. For unisexual sporulation, the wild-type strain XL280α and the cqs2Δ mutant were incubated on V8 medium in the presence or absence of Qsp1 for 7 days post inoculation. Scale bar: 20 μm. f, Deletion of CQS2 attenuated the expression of Cfl1-mCherry and synthetic Qsp1 cannot rescue this defect. Bars show mean ± SEM of four independent experiments. g, Overlap of Cqs2-regulated genes with Qsp1-regulated genes (n = 2 independent experiments). h, No Cqs2 targets showed differential expression in response to Qsp1 in the cqs2Δ mutant (n = 2 independent experiments). In c-e, images are representative of more than five independent experiments conducted with similar results.

Cqs2 protein does not possess any apparent domain except for a predicted nuclear localization signal (Fig. 6a). This is consistent with our observation that Cqs2-mCherry was enriched in the nucleus during unisexual mating (Fig. 6b). Upon further analysis in combining a Position-Specific Iterative BLAST analysis with Rosetta ab initio and comparative modeling methodology30, we identified a C2H2 zinc finger motif harbored in a 65-residue region that is highly conserved among proteins from phylogenetically divergent fungi (Fig. 6a,c and Supplementary Fig. 9). The C2H2 ZFs are the best-characterised class of zinc fingers to date and are extremely common in fungal transcription factors31. However, this conserved region of Cqs2 contains only one finger, and all characterised fungal C2H2 domains contain more than two. To examine the functional role of this “atypical” ZF motif in the unisexual induction activity of Cqs2, two cysteine residues predicted to be critical for zinc ion binding in the ZF motif were substituted with alanine to generate mutated Cqs2C416A/C421A. We found that mCherry-tagged Cqs2C416A/C421A localized normally and was expressed abundantly in cultured cells (Fig. 6b). However, forced expression of this mutated protein did not stimulate unisex in the cqs2Δ mutant, in which self-filamentation was substantially promoted as the wild-type version of Cqs2 was overexpressed (Fig. 6d).

Figure 6 |. Cqs2 can associate with its own promoter through an atypical C2H2 zinc finger domain conserved among divergent fungal species.

Figure 6 |

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a, Domain organization of Cqs2. NLS: nuclear localization signal. b, Localization of Cqs2-mCherry and Cqs2C416A/C421A-mCherry (> 120 cells for each were examined) during unisexual reproduction (top), and fluorescence intensity plot along a cellular axis indicated with a white line on the merged image (bottom). Scale bar: 5 μm. c, Phylogenetic tree of Cqs2 homologs. Protein sequences were aligned using neighbor-joining method with the MEGA v5.04 program. d, The CQS2C416A/C421A mutant allele cannot restore the defect of filamentation in the cqs2Δ mutant. Filamentation frequency (FF) was calculated based on the percentage of filamentous mini-colonies 21 hrs after mating induction. Data are presented as the mean ± SD from four independent experiments. e, ChIP was performed with anti-mCherry antibody in cqs1Δ mutant cells, in which the mCherry fused Cqs2 or Cqs2 C416A/C421A were overexpressed respectively. ChIP enrichment was detected by quantitative PCR across approximate 2 kb region upstream of or within the CQS2 ORF. Data shown is from two independent experiments.

To determine the capability of “atypical” C2H2 ZF protein Cqs2 to associate with DNA in vivo, we used chromatin immunoprecipitation (ChIP) assay to test whether it associates with its own promoter due to that many C2H2 members display auto-regulatory activities32. ChIP analysis was performed using anti-mCherry antibody (ChIP grade), and qPCR was used to evaluate the occupancy of Cqs2-mCherry at different locations upstream of or within the ORF of CQS2. We identified an evident enrichment of Cqs2-mCherry protein at the region surrounding the start site of the transcript of CQS2 identified by RNA-seq assay (Fig. 5b and Fig. 6e). The enrichment was significantly reduced in cells expressing Cqs2C416A/C421A-mCherry (Fig. 6e). These results indicate that Cqs2 associates with DNA, and specifically with its own promoter, and the ZF motif is important for its DNA association.

Discussion

In fungi, mating pheromone represents the best studied paracrine signal for sexual activation and it induces mating response through specific recognition by Ste2 or Ste3 type G protein-coupled receptors (GPCRs) upon external stimulation33, 34. Cryptococcus MATα cells harbor two Ste3 type GPCR genes: STE3α14 and CPR235. STE3α appears not to affect haploid fruiting14. By comparison, the expression of Cpr2 is important to induce unisex, but its activity for unisexual activation is constitutive and independent of ligands35. These evidence indicates that both GPCRs are unlikely to be involved in the direct recognition of secreted Mfα during unisexual mating, supporting the notion that the inability of Mfα to efficiently stimulate unisex in C. neoformans may be attributed to the lack of compatible GPCR receptor for its perception. We propose that such inability of the pheromone paracrine system may reflect the uniqueness of the unisexual cycle. Most eukaryotes, including Cryptococcus, engage in bisexual mating, in which nuclear fusion after cell-cell fusion (syngamy) between two compatible mating partners is the major mechanism involved in ploidy duplication before meiotic progression36. Previous research has indicated that unisex in C. neoformans can occur independently of syngamy8, 26, 37. Instead, endo-replication or cell fusion-independent karyogamy processes have been proposed as an alternative route to elevate ploidy8. Given that sexual syngamy is generally attributed to the control by pheromone in fungi38, unisex-specific syngamy-independent diploidization may provide a plausible explanation regarding why the intercellular regulation mediated by pheromone is not strictly required for unisexual reproduction in C. neoformans.

We show that the paracrine regulation exerted by QS molecule Qsp1 is important for both bisexual and unisexual reproduction in C. neoformans. Notably, cell density-dependent regulation of sexual reproduction, which is potentially mediated by QS-like molecule, has also been observed in other fungal species39, 40. For instance, in Candida albicans, the mating efficiency has been found to be positively linked with inoculum size when cells are grown anaerobically40. Because the Qsp1 system is not present in C. albicans, the effect of cell-density dependent control on sexual regulation may have arisen via convergent evolution. In bacteria, the QS system commonly coordinates gene expression in response to nutritional or environmental stressors. Early studies implicated that QS may have the same function in some fungi. For instance, in budding yeast, QS molecules phenylethanol and tryptophol have been found to control morphogenesis under nitrogen starvation conditions41. We also demonstrate that Qsp1 can stimulate mating response in response to glucose starvation, an important nutritional signal for many fungi. Nutritional and environmental stress has been broadly demonstrated in fungi to be involved in sexual initiation42, 43. In this regard, the QS system may play a general role as an intercellular circuit in bridging sexual control and perception of surrounding stress in fungi or other eukaryotic microbes.

Methods

Strains, culture conditions, mating and phenotypic assays

Strains used in this study are listed in Supplementary Table 6. All mutant strains were generated in the reference strains XL280α, a model C. neoformans strain used to investigate unisexual reproduction17, or JEC21α, which can undergo unisex, but less robustly than the former. Cryptococcus strains were cultured on YPD solid plates (20 g/l glucose, 20 g/l peptone, 10 g/l yeast extract, 2% agar) at 30°C for routine growth and V8 agar (5% v/v V8 juice, 0.5 g/l KH2PO4, 4% agar) for mating assays, unless otherwise indicated. For bilateral mating assays, a and α cells in equal numbers (original OD600 = 1.0) were premixed and co-cultured on V8 juice agar in the dark at 25°C. For quantitative analysis of filamentation frequency, cells of strains were plated onto V8 medium at a low cell density and allowed to grow into isolated mini-colonies after one day of culture. Mini-colonies exhibited a remarkable heterogeneity in filamentation, and the filamentous incidence among mini-colonies reflects the strength of unisexual induction. For carbon source-induced morphogenesis assays, YP medium (20 g/l peptone, 10 g/l yeast extract, 2% agar) without glucose was used as the base medium, to which a given carbon source was added to a final concentration of 2% (w/v). Strains carrying genes driven by the inducible promoter of the CTR4 gene were grown in medium supplemented with 25 μM of CuSO4 for suppression or 200 μM of bathocuproine disulphonate (BCS) for induction. Self-filamentation and sporulation were examined microscopically as previously described25. Since Qsp1 is highly diffusible, plates were cut to separate the cqs1Δ mutant from other strains harboring the intact CQS1 gene before the examination of phenotypes, unless otherwise indicated. All peptides used for mating and phenotypic assays were synthesized by Shanghai Qiangyao Bio-technology Co. Ltd.

Gene knockout and overexpression

For gene disruption, overlap PCR products were generated with NEO (Neomycin)/NAT (Nourseothricin) resistance cassette and 5’ and 3’ flanking sequences (1.2~1.5 kb) of the coding genes from strain XL280α as previously described26. The PCR product was directly introduced into strain XL280α by biolistic transformation. The resultant mutants were confirmed by PCR and genetic crosses. For gene overexpression, genes (ORF) were amplified by PCR and the amplified fragments were digested with proper restrictive digestion enzymes and inserted into pXC after the CTR4 promoter as previously described26. Overexpression plasmids were linearized via restriction enzyme digestion before being introduced into relevant Cryptococcus strains through biolistic transformation or electroporation as we previously described. Primers for gene disruption and overexpression used in this study are listed in Supplementary Table 7.

Microscopy and fluorescence

To examine the sub-cellular localization of Cqs2 during unisexual reproduction, strains harboring PCTR4-CQS2-mCherry were grown on V8 agar containing BCS at 25°C for 48 hrs. Five mg/ml DAPI was used for nucleus staining before being examined microscopically. To investigate the effect of CQS1 on the expression of Dmc1, cells from different strains harboring PDMC1-DMC1-mCherry were dropped on V8 agar plate and incubated at 25°C for 7 days. Images were acquired and processed with a Zeiss Axioplan 2 imaging system with the AxioCam MRm camera software Zen 2011 (Carl Zeiss Microscopy).

Confrontation analysis

The confrontation assay was used to test whether secreted factors from the wild-type strain can induce unisexual mating in Cryptococcus strains. 3 μl (OD600 = 3.0) of the donor strain cell suspension was dropped onto V8 plate and incubated in dark for about 72 hours at 25°C for the accumulation of the paracrine signal. Then 3μl of the low cell density recipient cells (OD600 = 0.05 - 0.1) were dropped in close proximity to the donor strain. Filamentation frequency was calculated based on the percentage of filamentous mini-colonies of the recipient strain.

Site-directed mutagenesis

We introduced single nucleotide mutations into CQS2 coding region to generate the mutant allele encoding Cqs2C416A/421A using the Quick Change II XL site directed mutagenesis kit (Agilent Technologies). Primers for mutagenesis (Supplementary Table 7) were designed using the QuickChange primer design program (Agilent Technologies).

RNA purification and RT-PCR analyses

Frozen pellets were ground in liquid nitrogen and total RNA was purified using the Ultrapure RNA Kit (Kangweishiji, CW0581S) according to the manufacturer’s instructions, and then reverse-transcribed with the Fastquant RT Kit (Tiangen KR106-02, with gDNase). Relative expression level of selected genes was measured by real time (RT)-PCR using power SYBR qPCR premix reagents (KAPA) in a CFX96 Touch™ Real-time PCR detection System (Biorad). Primers for qPCR used in this study are listed in Supplementary Table 7. Relative transcript levels were calculated to fold change and normalized to the TEF1 gene using the comparative CT method as previously described26.

RNA-seq and data analysis

For RNA-seq analysis evaluating the impact of Qsp1 signalling components on mating response, the wild-type XL280α strain and isogenic Qsp1 pathway mutants were cultured in YPD liquid medium (extremely mating-inhibitory condition) at 30°C overnight. The overnight cell culture was then washed by cold water, and plotted on V8 agar (pH = 7) with or without synthetic peptide or YP plates containing different carbon sources for mating induction. Cells were collected at 12 hrs post mating stimulation for isolation of total RNA, unless otherwise indicated. RNA levels and integrity were determined by Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologeis, CA, USA), respectively. RNA purity was evaluated using the Nano Photometer® spectrophotometer (IMPLEN, CA, USA). Illumina cDNA libraries were generated using VAHTS™ mRNA-seq v2 Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) according to the manufacturer’s instructions. Samples were clustered using VAHRS RNA adapters set1/set2 and sequenced using Illunima Hiseq 4000 platform in a 2 × 150 pair-ended manner.

Initial quality control of the sequencing data was performed using FastQC v0.11.5 software. After Initial quality control, Hisat v0.1.6-beta was used for clean short reads (~2 GB clean data for each sample, representing over 100 × coverage of the total transcriptome) mapping to the annotated genome sequence of Cryptococcus neoformans JEC21. The gene expression level was measured in FPKM (fragments per kilobase of exon per million fragments mapped) by Stringtie v1.2.1. The differential expression of genes (DEGs) was assessed using the Gfold v1.1.2 program44. Twenty-seven DEGs were chosen for quantitative real-time-PCR to confirm the reproducibility of the RNA-seq results. The genes with significant differential expression or significant transcriptional response to synthesized Qsp1 (Fig. 3e) were defined on the basis of the fold change criterion (|log2(fold change)| > 0.9). This criterion was determined according to gene functional grouping analysis, which enables precise assessment of biological significance of a given fold-change. DAVID gene ontology program (https://david.ncifcrf.gov/) was performed for gene ontology analysis. The heat maps were generated using the pheatmap package in R version 3.4.2.

Agrobacterium tumefaciens mediated random insertional mutagenesis (AIM) and AIM-sequencing

Random insertion mutagenesis was carried out as previously described using A. tumefaciens strain EHA105 carrying the PZP-NEO/NATcc plasmid with small modifications. Briefly, A. tumefaciens was incubated overnight with shaking at 28°C in Luria–Bertani (LB) broth containing kanamycin. Then the overnight A. tumefaciens cells were washed and resuspended at an OD660nm of 0.15 in liquid induction medium (IM) with 200 μM acetosyringone (AS), shaking at 22°C for 6 hrs, or until the final concentration reached an OD660nm of 0.6. An overnight culture of C. neoformans XL280α was collected and diluted in IM to a final concentration of 106 to 107 cells/ml. Equal volumes (200 μl) of A. tumefaciens and C. neoformans cells were then mixed, dropped without spreading on IM agar (with AS) and co-incubated at 22°C for 2-3 days before being scraped onto selection media (YPD + 100 μg/ml nourseothricin + 100 μg/ml cefotaxime). Colonies were then transferred into YPD liquid media and incubated at 30°C for 2–3 days before freezing down and/or screening.

For AIM-sequencing, the genomic DNA of the AIM mutants identified were pooled together with equal concentration to a final amount of 14.4 μg before whole genome sequencing using Illumina Hiseq 4000 (2 × 150bp pair-ended reads). The resulting sequence data contained 1.2×106 read pairs (~10 × genomic coverage per strain). The AIM-Seq analysis was performed online at https://github.com/granek/aimhii. A reference genome sequence, DNA sequence inserted, adapter sequences, and reads data sequenced in FASTQ format (either single-end or paired-end) are needed for AIM-HII analysis. The appropriate adapter sequence was supplied by Illumina. AIM-HII pairs the clusters that are within specified gap limit and flank opposite ends of the insert into “cluster pairs”. We refer to those unpaired clusters which have no partner identified as “singleton clusters”.

Chromatin Immunoprecipitation

ChIP assays were performed, as described previously with modifications45. In brief, cells incubated on V8 plates were harvested at 8 hrs post inoculation, and were cross-linked with 1% formaldehyde for 15 min. The crosslink was subsequently quenched with glycine at a final concentration of 125 mM for 5 min. Chromatin fragmentation was achieved using Microccocal Nuclease (New England BioLabs) digestion for 15 min at 37°C. 2 U Microccocal nuclease was used for samples containing per mg total protein. Nuclear membrane was then disrupted by sonication (Diagenode Biorupter) at 4°C. Clarified chromatin extracts were incubated with ChIP grade anti-mCherry antibody (Chromo Tek, RFP-TRAP, rtma-20, coupled to magnetic beads) overnight with agitation. After stringent washes and elution, elutes were reverse cross-linked and de-proteinated with NaCl and protease K at 65°C for 4 hrs. Then, DNA was extracted using phenol-chloroform, followed by ethanol precipitation with glycan. ChIP-qPCR assays were performed with primers at different locations upstream of or within CQS2 ORF. The enrichment of the signal was quantified as percentage of input for each primer pair. All primers used in ChIP-qPCR analyses are shown in Supplementary Table 7.

Statistical analysis

Statistical analyses were performed with R, version 3.4.2. We used a two-tailed unpaired Student t test to compare the mean florescence intensity or transcript levels from two groups. For all analysis, P values < 0.05 were considered significant and P values < 0.001 were considered very significant. Error bars reflect means ± SD or means ± SEM from three or more independent experiments.

Life Sciences Reporting Summary

Further information on experimental design is available in the Life Sciences Reporting Summary.

Supplementary Material

ST1
SF1-9 and ST3
ST2
ST4
ST6
ST5
ST7

Acknowledgements

We thank Prakriti Sharma and Wang lab members for critical reading, and Dr. Shuai Luo and Ms. Huimin Liu for their assistance in genetic screening. This work was financially supported by the Key Research Program of the Chinese Academy of Sciences (QYZDB-SSW-SSMC040), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDPB03), National Natural Science Foundation of China (Grants 31622004, 31570138, 31501008 and 31501009) and National Institutes of Health (http://www.niaid.nih.gov/Pages/default.aspx) (R01AI097599 to XL). Linqi Wang is a member of the “Thousand Talents Program”. Xiaorong Lin holds an Investigator Award in the Pathogenesis of Infectious Disease from the Burroughs Welcome Fund (http://www.bwfund.org/).

Footnotes

Competing interests

The authors declare no competing financial interests.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The GEO accession number for the RNA-seq data reported in this study is GSE94091.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ST1
NIHMS2137067-supplement-ST1.xlsx (429.9KB, xlsx)
SF1-9 and ST3
NIHMS2137067-supplement-SF1-9_and_ST3.docx (2.5MB, docx)
ST2
NIHMS2137067-supplement-ST2.xlsx (1.4MB, xlsx)
ST4
NIHMS2137067-supplement-ST4.xlsx (188.6KB, xlsx)
ST6
NIHMS2137067-supplement-ST6.xlsx (12.2KB, xlsx)
ST5
NIHMS2137067-supplement-ST5.xlsx (238.2KB, xlsx)
ST7
NIHMS2137067-supplement-ST7.xlsx (13.1KB, xlsx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon request. The GEO accession number for the RNA-seq data reported in this study is GSE94091.

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