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Hyphal growth is strongly associated with virulence in the human fungal pathogenCandida albicans. While hyphal transcriptional networks have been the subject of intense study, relatively little is known about post-transcriptional regulation. Previous work reported that P Body (PB) factors Dhh1 and Edc3 were required for virulence and filamentation, suggesting an essential role for post-transcriptional regulation of these processes. However, the molecular roles of these factors have not been determined. To further study the function of PB factors in filamentation, we generated homozygous deletions ofDHH1andEDC3in the prototrophic strain SC5314 using CRISPR-Cas9. Homozygousdhh1deletion strongly impaired growth and altered filamentation, in addition to exhibiting unusual colony morphology in response to heat stress.Using RNA-seq, we foundDHH1deletion disrupts the regulation of thousands of genes under both yeast and hyphal growth conditions. This included upregulation of many stress response genes in the absence of stress, similar to deletion of theS. cerevisiae DHH1homolog. In contrast, we foundEDC3was not required for heat tolerance or filamentation. These results support a model in whichDHH1, but notEDC3, represses hyphal stress response transcripts in yeast and remodels the transcriptome during filamentation. Our paper supports distinct requirements for specific mRNA decay factors, bolstering evidence for post-transcriptional regulation of filamentation inC. albicans.Author summaryIn the dimorphic fungal pathogenC. albicans, the hyphal phenotype corresponds with pathogenicity. While transcriptional control of hyphal growth has been extensively studied, comparatively little is known about post-transcriptional regulation of this significant morphological shift. PB factors are associated with mRNA decay and translational repression. Here we investigate the roles of two PB factors in growth, filamentation, and gene expression. Although deletion of PB factor EDC3 did not impact growth or filamentation,dhh1Δ/Δhad greatly impaired growth and heat tolerance as well as unusual hyphal phenotypes. Additionally, we found that the transcriptomes ofdhh1Δ/Δyeast and hyphae were highly dysregulated. The extensive transcriptomic impacts of the absence of Dhh1 correlated with our phenotypic findings. Stress-associated genes were induced under non-stress conditions and the filamentation response was blunted under physiologically relevantin vitroconditions. We demonstrate that mRNA decay factors play distinct roles in regulatingC. albicansmorphology and that Dhh1 contributes to environmentally appropriate expression of the stress response and hyphal growth.
Hyphal growth is strongly associated with virulence in the human fungal pathogenCandida albicans. While hyphal transcriptional networks have been the subject of intense study, relatively little is known about post-transcriptional regulation. Previous work reported that P Body (PB) factors Dhh1 and Edc3 were required for virulence and filamentation, suggesting an essential role for post-transcriptional regulation of these processes. However, the molecular roles of these factors have not been determined. To further study the function of PB factors in filamentation, we generated homozygous deletions ofDHH1andEDC3in the prototrophic strain SC5314 using CRISPR-Cas9. Homozygousdhh1deletion strongly impaired growth and altered filamentation, in addition to exhibiting unusual colony morphology in response to heat stress.Using RNA-seq, we foundDHH1deletion disrupts the regulation of thousands of genes under both yeast and hyphal growth conditions. This included upregulation of many stress response genes in the absence of stress, similar to deletion of theS. cerevisiae DHH1homolog. In contrast, we foundEDC3was not required for heat tolerance or filamentation. These results support a model in whichDHH1, but notEDC3, represses hyphal stress response transcripts in yeast and remodels the transcriptome during filamentation. Our paper supports distinct requirements for specific mRNA decay factors, bolstering evidence for post-transcriptional regulation of filamentation inC. albicans.Author summaryIn the dimorphic fungal pathogenC. albicans, the hyphal phenotype corresponds with pathogenicity. While transcriptional control of hyphal growth has been extensively studied, comparatively little is known about post-transcriptional regulation of this significant morphological shift. PB factors are associated with mRNA decay and translational repression. Here we investigate the roles of two PB factors in growth, filamentation, and gene expression. Although deletion of PB factor EDC3 did not impact growth or filamentation,dhh1Δ/Δhad greatly impaired growth and heat tolerance as well as unusual hyphal phenotypes. Additionally, we found that the transcriptomes ofdhh1Δ/Δyeast and hyphae were highly dysregulated. The extensive transcriptomic impacts of the absence of Dhh1 correlated with our phenotypic findings. Stress-associated genes were induced under non-stress conditions and the filamentation response was blunted under physiologically relevantin vitroconditions. We demonstrate that mRNA decay factors play distinct roles in regulatingC. albicansmorphology and that Dhh1 contributes to environmentally appropriate expression of the stress response and hyphal growth.
Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans . An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases. IMPORTANCE Candida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.
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