Strain variation in gene expression impact of hyphal cyclin Hgc1 in Candida albicans

Abstract Formation of hyphae is a key virulence trait of the fungal pathogen Candida albicans. Hypha morphogenesis depends upon the cyclin Hgc1, which acts together with cyclin-dependent protein kinase Cdc28 to phosphorylate effectors that drive polarized growth. Hgc1 has also been implicated in gene regulation through its effects on 2 transcription factors, Efg1 and Ume6. Here, we report RNA-sequencing (RNA-seq) analysis of 2 pairs of hgc1Δ/Δ mutants and their respective wild-type strains, which lie in 2 different genetic backgrounds. We find that hgc1Δ/Δ mutations alter expression of 271 genes in both genetic backgrounds and 266 of those genes respond consistently with regard to up- or down-regulation. Consistency is similar to what has been observed with efg1Δ/Δ mutations and greater than observed with nrg1Δ/Δ mutations in these 2 backgrounds. The gene expression response includes genes under Efg1 control, as expected from prior studies. Hgc1-responsive genes also include ergosterol biosynthetic genes and bud neck-related genes, which may reflect interactions between Hgc1 and additional transcription factors as well as effects of Hgc1 on cellular length-to-width ratios.


Introduction
Candida albicans is a commensal fungus that causes mucosal and lethal invasive infections in at-risk individuals (Polvi et al. 2015).One of the most well-studied virulence traits of this organism is its ability to transition from the yeast form to filamentous hyphae (Sudbery 2011).Hyphae are noteworthy both for their long tubular morphology and for their high-level expression of a set of hypha-associated genes (Mayer et al. 2013;Wilson et al. 2016;Chow et al. 2021).These genes specify multiple functions, including a panel of regulators that reinforce and modulate the hyphal gene expression program; an arsenal of virulence effectors that include adhesins and invasins, the Candidalysin toxin, diverse hydrolases, and neutrophil defense functions; and the hyphal-specific G1 cyclin 1 (HGC1) gene product, a hypha-specific cyclin (Mayer et al. 2013;Wang 2016;Wilson et al. 2016;Rodriguez et al. 2020).
HGC1 is expressed constitutively and independently of the cell cycle in hyphal cells (Zheng et al. 2004).Hgc1 partners with the cyclin-dependent kinase (CDK) Cdc28 to phosphorylate numerous effector proteins, thus promoting polarized growth at the hyphal tip (Zheng et al. 2004;Wang 2016;Chow et al. 2021).This role of Hgc1 has been extremely well established through phosphorylation assays, target site mutations, and other approaches (Wang 2016;Chow et al. 2021).However, a second role of Hgc1 in gene regulation is less well understood.Hgc1 has been shown to affect 2 transcription factors that promote hypha formation, Efg1 and Ume6.Efg1 is phosphorylated by the Hgc1-Cdc28 complex (Wang et al. 2009).Phosphorylation increases Efg1 binding to promoters of cell separation genes, such as CHT3, SCW11, and DSE1, that are activated by the transcription factor Ace2 (Wang et al. 2009).The relationship between Ume6 and Hgc1 is more complex, because Hgc1 has 2 characterized functions: to promote Ume6 degradation and to stimulate translation of the UME6 mRNA (Mendelsohn et al. 2017).Therefore, Hgc1 affects the levels or activity of at least 2 transcriptional regulators.
Although Hgc1 affects Efg1 and Ume6, previous studies have detected little if any effect of an hgc1Δ/Δ mutation on the expression of hypha-associated genes (Zheng et al. 2007;Chao et al. 2010;Wang 2016;Chow et al. 2021).These studies used state-of-the-art methods at the time-Northern blot analysis and qRT-PCR-but were limited by sensitivity and the use of gene-targeted strategies.In addition, the studies addressed gene expression impact only in derivatives of the C. albicans-type strain SC5314.Reliance on a type strain has been vital for progress in our understanding of C. albicans (see for example Noble and Johnson 2007;Crunden and Diezmann 2021;Wang et al. 2021), but comparisons among strains can indicate which gene expression features have greatest functional significance (Morschhäuser et al. 2007;Huang et al. 2019;Wang et al. 2021;Do et al. 2022;Cravener et al. 2023).Here, we present genome-wide expression profiles of hgc1Δ/Δ mutants in 2 different C. albicans genetic backgrounds.Our findings indicate that the scope of Hgc1 gene expression impact is maintained in both strains, though the magnitude varies, in keeping with differences in phenotypic impact we recently reported (Sharma et al. 2023).

Media
All strains used in the study are listed in Supplementary Table 1.They were maintained in 15% glycerol stocks stored at −80°C.Before all experiments, strains were grown on Yeast extract-Peptone-Dextrose (YPD) (2% Bacto Peptone, 2% dextrose, and 1% yeast extract) for 2 days at 30°C.For RNA-sequencing (RNA-seq) experiments, cells were grown in RPMI 1640 medium (Sigma-Aldrich), adjusted to pH 7.4 and supplemented with 10% fetal bovine serum (Atlanta Biologicals).

RNA extraction and NanoString
RNA extractions and NanoString analysis were done according to the previously described method (Woolford et al. 2016).Briefly, RNA extraction was performed using a Qiagen RNeasy mini kit (Cat#74104) with some modifications.Then, samples were loaded on nCounter SPRINT cartridge for scanning on an nCounter digital analyzer.Raw counts were normalized against average total counts with background subtraction.

RNA sequencing
RNA was extracted from the mentioned strains using a Qiagen RNeasy mini kit (Cat#74104) according to the previously described method with some modifications (Woolford et al. 2016).A total amount of 1-μg RNA per sample was used as input material for the RNA-seq sample preparations.Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample.To select cDNA fragments of preferentially 150-200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA).Then, 3-μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR.Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer.At last, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions.After cluster generation, the library preparations were sequenced on an Illumina platform, and 125-/150-bp paired-end reads were generated.The index of the C. albicans reference genome (Assembly A21) was built using hisat2 2.1.0,and paired-end clean reads were aligned to the reference genome using HISAT2.FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene.And then, the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of each gene was calculated based on the length of the gene and the reads count mapped to this gene.Differential expression analysis between 2 conditions/groups (3 biological replicates per condition) was performed using the DESeq2 R package (1.14.1).Genes with an adjusted P-value < 0.05 found by DESeq2 were assigned as differentially expressed.Pearson correlations between replicates were in the range of 0.952-0.998,while Pearson correlations of WT vs mutant comparisons were in the range 0.921-0.986(Supplementary File 2, "Sample correlations" tab).

Software
Gene ontology (GO) term enrichment was performed using the FungiFun online tool.Venn diagram was made using online tool Draw Venn Diagram (ugent.be).

Impact of Hgc1 on hypha-associated gene expression
We used NanoString assays for an initial determination of the effect of an hgc1Δ/Δ mutation on hypha-associated gene expression (Supplementary File 1).We included hgc1Δ/Δ mutants of 5 clinical isolates: SC5314 (clade 1), P76067 (clade 2), P57055 (clade 3), GC75 (clade 4), and 19F (clade 1).Cells were grown under strong hypha-inducing conditions (RPMI + serum at 37°C for 4 h).One hundred sixty genes were included in the assay, many of which are associated with hyphal growth and biofilm formation (Huang et al. 2019).The results suggested that several genes were differentially expressed in each mutant compared with its respective wild type, including hypha-associated genes SAP5, HYR1, SAP6, HWP1, and ECE1 (Supplementary Fig. 1).More severe gene expression defects were observed in genetic backgrounds with more severe morphogenesis defects (Sharma et al. 2023).The results also suggested that the magnitude of gene expression differences between mutant and wild type may vary with strain background.
We pursued these findings through RNA-seq analysis of the SC5314 and P57055 strain backgrounds after hyphal induction in RPMI + serum at 37°C for 4 h (Supplementary File 2).Gene expression impact of the hgc1Δ/Δ mutation varied considerably between the 2 backgrounds (Fig. 1a), based on conventional thresholds of an adjusted P-value < 0.05 and a fold change > 2 (Supplementary File 2, DEG tabs).In SC5314, 38 genes had altered expression in the hgc1Δ/Δ mutant.In P57055, 120 genes had altered expression in the hgc1Δ/Δ mutant.Only 17 genes responded significantly in both strain backgrounds based on these cutoffs (Fig. 1a).The outcomes mirrored previous findings regarding strain variation in the impact of transcription factor gene mutations (Morschhäuser et al. 2007;Huang et al. 2019;Do et al. 2022;Cravener et al. 2023;Mao et al. 2023).
Many genes showed a similar trend in both strain backgrounds, but fold-change values were less in strain SC5314 than in strain P57055.This difference in gene expression may underly the more modest severity of the hgc1Δ/Δ morphogenesis defect in strain SC5314 than in strain P57055 (Sharma et al. 2023).To determine whether there may be underlying uniform impact of hgc1Δ/Δ mutations, we compared genes with statistically significant expression changes (adjusted P-value < 0.05) in the 2 strain backgrounds regardless of their fold change (Supplementary File 2, fold-change comparison tab).There were 271 genes in that group A. Sharma and A. P. Mitchell | 3 and 266 of the genes responded similarly (up-regulated or downregulated) to hgc1Δ/Δ mutations in both strains.The results for the 2 strain backgrounds correlated with an R 2 = 0.75 (Fig. 1b).The most significantly enriched GO terms for the genes with correlated responses included cell surface, plasma membrane, ergosterol biosynthesis, and cell wall (Fig. 2).These categories describe down-regulated genes for the most part (Fig. 2 and Supplementary File 3).Especially noteworthy was the down-regulation of many ergosterol biosynthetic genes in the hgc1Δ/Δ mutants of both strains (Fig. 2c).Overall, the effects of hgc1Δ/Δ mutations on gene expression align with the effects on hypha formation, in that the mutant defect is evident in both strains but less severe in SC5314 than in P57055 (Sharma et al. 2023).
How strong is the correlation between Hgc1-responsive genes when only statistical significance, not fold change, is used as a cutoff?To address this question, we examined data sets for 2 published mutant vs wild-type RNA-seq comparisons that were carried out with the same 2 strains backgrounds, SC5314 and P57055.One study examined efg1Δ/Δ mutants of both strains (Cravener et al. 2023).From those data sets, Efg1-responsive gene expression changes affected 2,083 genes and correlated with an R 2 = 0.79 between the strains (Fig. 1c).A second study examined nrg1Δ/Δ mutants of both strains (Mao et al. 2023).From those data sets, Nrg1-responsive gene expression changes affected 1,848 genes and correlated with an R 2 = 0.38 between the strains (Fig. 1c).Therefore, Hgc1-responsive gene expression changes correlate about as well as Efg1-responsive gene expression changes and considerably better that Nrg1-responsive gene expression changes.
How may Hgc1 influence gene expression?Work from Wang et al. (2009) showed that the Hgc1-Cdc28 complex phosphorylates transcription factor Efg1, causing it to antagonize the transcriptional activator Ace2.Their mutant analysis indicated that phospho-Efg1 represses Ace2-regulated cell separation genes, including CHT3, SCW11, DSE1, and PGA38.Three of these genes-CHT3, SCW11, and PGA38-are among the up-regulated genes that we found in hgc1Δ/Δ mutants.This relationship prompted us to make a broader comparison of Hgc1-responsive genes and Ace2 target genes.Among 271 Hgc1-responsive genes, we found 12 to 22 Ace2-regulated genes that had been identified in the studies of van Wijlick et al. (2016) and Desai et al. (2015).This comparison suggests that the impact of Hgc1 extends beyond the Ace2 regulatory circuit.Does Hgc1 act primarily through interaction with Efg1?Among 271 Hgc1-responsive genes identified, 113 genes have 5′ regions that are bound by Efg1 in multiple strains (Do et al. 2022); only 25 of the 113 genes show Efg1-dependent expression changes in multiple backgrounds (Cravener et al. 2023).Therefore, our results are not only consistent with the established role of Hgc1 in stimulating Efg1-DNA binding (Wang et al. 2009;Wang 2016) but also argue that Hgc1 affects gene expression independently of Efg1.
To our knowledge, the results presented here are the first genome-wide analysis of Hgc1-responsive gene expression.The effect of an hgc1Δ/Δ mutation on gene expression under our growth conditions is modest in magnitude but relatively consistent between 2 genetic backgrounds in scope.We note that we have used only 1 growth medium, temperature, and time point for gene expression assays, and expression differences may vary qualitatively or quantitatively under other conditions.Hgc1-responsive genes include some under Ace2 or Efg1 control, as expected from prior mechanistic studies.Hgc1-responsive genes also include some surprises, such as ergosterol biosynthetic genes and bud neck-related genes, that may reflect interactions between Hgc1 and additional transcription factors.Our findings help to illuminate the breadth of roles of Hgc1 in C. albicans biology.

Fig. 1 .
Fig.1.Gene expression analysis of hgc1Δ/Δ mutants.a) Global expression was assayed using RNA-seq.Three biological replicates were analyzed for hgc1Δ/Δ mutant and wild type of SC5314 and P57055 clinical isolates from cells grown in RPMI + 10% serum for 4 h at 37°C.Venn diagrams depict the genes dependent upon HGC1 in both strain backgrounds with >2-fold expression change and P < 0.05.b) Regression analysis shows the number of significantly regulated (P < 0.05) HGC1-responsive genes, regardless of fold change.The X and Y axes represent hgc1Δ/Δ vs wild-type log fold change for SC5314 and P57055, respectively.The data sets correlate with R 2 = 0.74.c) Regression analysis shows the number of significantly regulated (P < 0.05) EFG1-responsive genes, regardless of fold change, from previously published data(Cravener et al. 2023).The X and Y axes represent efg1Δ/Δ vs wild-type log fold change for SC5314 and P57055, respectively.The data sets correlate with R 2 = 0.78.d) Regression analysis shows the number of significantly regulated (P < 0.05) NRG1-responsive genes, regardless of fold change, from previously published data(Mao et al. 2023).The X and Y axes represent nrg1Δ/Δ vs wild-type log fold change for SC5314 and P57055, respectively.The data sets correlate with R 2 = 0.38.

Fig. 2 .
Fig. 2. GO term analysis of Hgc1-regulated genes.a) The HGC1-responsive genes that were significantly regulated (P < 0.05) in hgc1 mutants of SC5314 and P57055 background were analyzed for GO term enrichment using online tool FungiFun.Pie chart showing the major GO term distribution of the genes.b) Table showing the gene names representing significant GO terms.c) Graph of log 2 fold change values for expression of ERG genes in hgc1Δ/Δ mutants vs WT of SC5314 (blue bars) and P57055 (red bars) backgrounds.An asterisk marks log fold-change values with a P adj value of <0.05 (Supplementary File 2, "RNA SEQ LOG2 FOLD CHANGE" tab).