The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmit external signals into the inside of the cell, triggering appropriate biological responses. The external signals are sensed by various G-protein-coupled receptors (GPCRs) and transmitted into G-proteins consisting of the α, β, and γ subunits. Regulators of G-protein signaling (RGSs) are the key controllers of G-protein signaling pathways. GPCRs, G-proteins, and RGSs are the primary upstream components of the G-protein signaling pathway, and they are highly conserved in most filamentous fungi, playing diverse roles in biological processes. Recent studies characterized the G-protein signaling components in the opportunistic pathogenic fungus Aspergillus fumigatus. In this review, we have summarized the characteristics and functions of GPCRs, G-proteins, and RGSs, and their regulatory roles in governing fungal growth, asexual development, germination, stress tolerance, and virulence in A. fumigatus.

The velvet regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus Aspergillus nidulans. In the present study, we characterize the roles of VosA in sexual spores (ascospores) in A. nidulans. During ascospore maturation, the deletion of vosA causes a rapid decrease in spore viability. The absence of vosA also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of vosA causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of vosA results in the downregulation of various genes involved in primary metabolism. In addition, vosA deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the velvet regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in A. nidulans.

The calcineurin pathway is an important signaling cascade for growth, sexual development, stress response, and pathogenicity in fungi. In this study, we investigated the function of CrzA, a key transcription factor of the calcineurin pathway, in an aflatoxin-producing fungus Aspergillus flavus (A. flavus). To examine the role of the crzA gene, crzA deletion mutant strains in A. flavus were constructed and their phenotypes, including fungal growth, spore formation, and sclerotial formation, were examined. Absence of crzA results in decreased colony growth, the number of conidia, and sclerocia production. The crzA-deficient mutant strains were more susceptible to osmotic pressure and cell wall stress than control or complemented strains. Moreover, deletion of crzA results in a reduction in aflatoxin production. Taken together, these results demonstrate that CrzA is important for differentiation and mycotoxin production in A. flavus.

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.

Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1 , established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1 Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1 Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1 Δ/Δ mutation is most prominent under static or “biofilm-like” growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1 : hypha-associated gene expression levels are substantially reduced in a brg1 Δ/Δ mutant and partially restored in a brg1 Δ/Δ rme1 Δ/Δ double mutant. An rme1 Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions. IMPORTANCE: Candida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1 , whose activity is most prominent under biofilm-like growth conditions.

Central carbon metabolism is vital for the proliferation of Candida albicans , a fungus that is prominent as a commensal and pathogen. Glycolytic genes are activated by overlapping activities of the transcription factors Tye7 and Gal4, as shown by studies in the SC5314 genetic background. However, regulatory relationships can vary among C. albicans isolates. Here, we analyzed Tye7- and Gal4-related phenotypes in five diverse clinical isolates of C. albicans . We tested growth properties and gene expression impact through Nanostring profiling and, for the two strains SC5314 and P87, RNA sequencing. Our results lead to three main conclusions. First, the functional redundancy of Tye7 and Gal4 for glycolytic gene activation is preserved among all strains tested. Second, at the gene expression level, strain P87 is an outlier with regard to tye7 Δ/Δ impact, and strain SC5314 is an outlier with regard to gal4 Δ/Δ impact. Third, while Gal4 is well known to be dispensable for induction of the GAL1 , GAL7 , and GAL10 galactose-specific metabolic genes, we find that gal4 Δ/Δ mutants of several strains have a mild galactose fermentation defect, as assayed by growth on galactose with the respiration inhibitor antimycin A. Our findings indicate that even a central metabolic regulatory network is subject to strain variation and illustrates an unexpected genotype-phenotype relationship. The fungal commensal and pathogen Candida albicans rely upon metabolic flexibility to colonize and infect host niches. Central carbon metabolism is governed by two regulators, Tye7 and Gal4, as defined in the reference strain SC5314. Here, we have explored the impact of Tye7 and Gal4 on carbon utilization and gene expression across five diverse C. albicans clinical isolates. Novel aspects of this study are the finding that even a central metabolic regulatory network is subject to strain variation and the observation of an unexpected mutant phenotype.