Hydroxyurea Enhances Post-Fusion Hyphal Extension During Sexual Development in C. neoformans var. grubii

Abstract

Mating and sexual development in C. neoformans var. grubii strains of the H99 background is often less robust than that laboratory generated isogenic C. neoformans var. neoformans strains in the JEC21 background. In Candida albicans and Saccharomyces serevisiae, slowing of DNA synthesis and engagement of the replication stress response, such as that caused by treatment with hydroxyurea (HU), induces filamentation and pseudohyphal growth, respectively. In this study, we investigated the effect of HU treatment on C. neoformans var. grubii morphogenesis. Treatment with HU did not induce filamentation of yeast cells either in liquid culture or on solid YPD or V8 agar. In the presence of the opposite mating partner, we observed early emergence of hyphae in the presence of HU. Semi-quantitative analysis of fusion using marked strains demonstrated that no significant enhancement of fusion in the presence of HU. Transfer of fusion colonies from crosses performed in the absence of HU to V8 + HU revealed enhanced hyphal growth in the presence of HU. Analysis of expression of the target of HU, ribonucleotide reductase, revealed that a phylogenetically divergent catalytic subunit is replication stress responsive in C. neoformans. These results suggest that induction of replication stress promotes post-fusion hyphal growth of C. neoformans var. grubii strains in the H99 background.

The cellular roles of Ccr4-NOT in model and pathogenic fungi—implications for fungal virulence

“…C. neoformans lacks an ortholog of CRT1 , and induces a basidiomycete-specific ribonucleotide reductase small subunit in response to HU treatment, rather than the retained ascomycete-like gene (Zulkifli et al, 2012). An effect of CCR4 deletion on the regulation of these genes has yet to be determined.…”

Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans

“…ScRNR genes, including RNR1 and RNR2 , and C. albicans RNR2 are essential for viability ( 3 , 4 , 18 ). A previous study suggested that C. neoformans has one RNR1 homolog, encoding a large RNR subunit, and two homologous genes, RNR21 and RNR22 , which encode small RNR subunits ( 19 ). To address whether CnRNR1 , CnRNR21 , and CnRNR22 are required for survival, we constructed conditional RNR1 , RNR21 , and RNR22 expression strains by replacing each native promoter with a copper-regulated CTR4 promoter ( P CTR4 ) upstream of the ATG start codon or the 5′-UTR of the RNR1 , RNR21 and RNR22 genes (Fig.…”

“…The hydroxyurea (HU), which is an RNR inhibitor causing depletion of dNTPs, is widely used for induction of DNA replication stress ( 2 ). Given that RNR1 and RNR21 expression is induced in C. neoformans and that the expression levels of RNR2 , RNR3 , and RNR4 are regulated by Rad53 kinases under DNA replication stress in S. cerevisiae ( 5 , 19 ), we measured the expression levels of RNR genes under HU treatment in the WT and rad53 Δ mutant strains. Unlike RNR expression patterns in S. cerevisiae , RNR1 and RNR21 induction levels in the rad53 Δ mutant were slightly lower than those in the WT ( Fig.…”

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen

“…Since Dnj4 plays a role in the response to DNA damage, we characterized the transcriptional response of the WT and the dnj4Δ deletion strains to HU, which depletes cellular levels of nucleotides and results in induction of the DNA damage stress responses ( 26 , 27 ). Transcriptome sequencing (RNA-Seq) was conducted on HU-treated and untreated cells of both strains.…”