Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens.

In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen Cryptococcus neoformans has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by HXL1 (HAC1 and XBP1-Like gene 1). C. neoformans HXL1 encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of HXL1 and its unconventional splicing is widely conserved in C. neoformans strains of divergent serotypes. Notably, both C. neoformans ire1 and hxl1 mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the ire1 mutant were suppressed by the spliced active form of Hxl1, supporting that HXL1 mRNA is a downstream target of Ire1. Interestingly, however, the ire1 and hxl1 mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in C. neoformans. Finally, Ire1 and Hxl1 were shown to be critical for virulence of C. neoformans, suggesting UPR signaling as a novel antifungal therapeutic target.

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets.

SummaryThe RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RNAdependent RNA polymerase (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. Comparative transcriptome analysis of the serotype A and D RNAi mutants revealed that only modest changes occur in the genome-wide transcriptome profiles when the RNAi process was perturbed. Notably, the serotype D rdp1Δ mutants showed an increase in transcript abundance of active retrotransposons and transposons, such as T2 and T3, the latter of which is a novel serotype D-specific transposon of C. neoformans. In a wild type background both T2 and T3 were found to be weakly active mobile elements, although we found no evidence of Cnl1 retrotransposon mobility. In contrast, all three transposable elements exhibited enhanced mobility in the rdp1Δ mutant strain. In conclusion, the RNAi pathway plays an important role in controlling transposon activity and genome integrity of C. neoformans.

The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1⌬, gpa1⌬, cac1⌬, aca1⌬, and pka1⌬ pka2⌬ mutants. The aca1⌬, gpa1⌬, cac1⌬, and pka1⌬ pka2⌬ mutants displayed similar transcriptome patterns, whereas the ras1⌬ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1⌬ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans.

The basidiomycetous fungus Cryptococcus neoformans has been known to be highly radiation resistant and has been found in fatal radioactive environments such as the damaged nuclear reactor at Chernobyl. To elucidate the mechanisms underlying the radiation resistance phenotype of C. neoformans, we identified genes affected by gamma radiation through genome-wide transcriptome analysis and characterized their functions. We found that genes involved in DNA damage repair systems were upregulated in response to gamma radiation. Particularly, deletion of recombinase RAD51 and two DNA-dependent ATPase genes, RAD54 and RDH54, increased cellular susceptibility to both gamma radiation and DNA-damaging agents. A variety of oxidative stress response genes were also upregulated. Among them, sulfiredoxin contributed to gamma radiation resistance in a peroxiredoxin/thioredoxin-independent manner. Furthermore, we found that genes involved in molecular chaperone expression, ubiquitination systems, and autophagy were induced, whereas genes involved in the biosynthesis of proteins and fatty acids/sterols were downregulated. Most importantly, we discovered a number of novel C. neoformans genes, the expression of which was modulated by gamma radiation exposure, and their deletion rendered cells susceptible to gamma radiation exposure, as well as DNA damage insults. Among these genes, we found that a unique transcription factor containing the basic leucine zipper domain, named Bdr1, served as a regulator of the gamma radiation resistance of C. neoformans by controlling expression of DNA repair genes, and its expression was regulated by the evolutionarily conserved DNA damage response protein kinase Rad53. Taken together, the current transcriptome and functional analyses contribute to the understanding of the unique molecular mechanism of the radiation-resistant fungus C. neoformans.

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress-response, and maintenance of cell wall integrity. Here we characterized a C. neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyperfilamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.

bProtein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1-and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets.