Pleiotropic roles of the Msi1-like protein Msl1 in Cryptococcus neoformans.

Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans. Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78. In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans, providing insight into a potential novel antifungal therapeutic target.

Comparative transcriptome analysis reveals novel roles of the Ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus 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 ras1Delta, gpa1Delta, cac1Delta, aca1Delta, and pka1Delta pka2Delta mutants. The aca1Delta, gpa1Delta, cac1Delta, and pka1Delta pka2Delta mutants displayed similar transcriptome patterns, whereas the ras1Delta 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 ras1Delta 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.

Characterizing the role of RNA silencing components in Cryptococcus neoformans.

The 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), RNA-dependent 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.

Regulatory Mechanism of the Atypical AP-1-Like Transcription Factor Yap1 in Cryptococcus neoformans.

AP-1-like transcription factors play evolutionarily conserved roles as redox sensors in eukaryotic oxidative stress responses. In this study, we aimed to elucidate the regulatory mechanism of an atypical yeast AP-1-like protein, Yap1, in the stress response and virulence of Cryptococcus neoformansYAP1 expression was induced and involved not only by oxidative stresses, such as H2O2 and diamide, but also by other environmental stresses, such as osmotic and membrane-destabilizing stresses. Yap1 was distributed throughout both the cytoplasm and the nucleus under basal conditions and more enriched within the nucleus in response to diamide but not to other stresses. Deletion of the C-terminal cysteine-rich domain (c-CRD), where the nuclear export signal resides, increased nuclear enrichment of Yap1 under basal conditions and altered resistance to oxidative stresses but did not affect the role of Yap1 in other stress responses and cellular functions. As a potential upstream regulator of Yap1, we discovered that Mpk1 is positively involved, but Hog1 is mostly dispensable. Pleiotropic roles for Yap1 in diverse biological processes were supported by transcriptome data showing that 162 genes are differentially regulated by Yap1, with further analysis revealing that Yap1 promotes cellular resistance to toxic cellular metabolites produced during glycolysis, such as methylglyoxal. Finally, we demonstrated that Yap1 plays a minor role in the survival of C. neoformans within hosts.IMPORTANCE The human meningitis fungal pathogen, Cryptococcus neoformans, contains the atypical yeast AP-1-like protein Yap1. Yap1 lacks an N-terminal cysteine-rich domain (n-CRD), which is present in other fungal Yap1 orthologs, but has a C-terminal cysteine-rich domain (c-CRD). However, the role of c-CRD and its regulatory mechanism remain unknown. Here, we report that Yap1 is transcriptionally regulated in response to oxidative, osmotic, and membrane-destabilizing stresses partly in an Mpk1-dependent manner, supporting its role in stress resistance. The c-CRD domain contributed to the role of Yap1 only in resistance to certain oxidative stresses and azole drugs but not in other cellular functions. Yap1 has a minor role in the survival of C. neoformans in a murine model of systemic cryptococcosis.

The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1.

In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to ß-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression.

Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human pathogenic fungus that causes meningoencephalitis. Due to the increasing global risk of cryptococcosis and the emergence of drug-resistant strains, the development of predictive genetics platforms for the rapid identification of novel genes governing pathogenicity and drug resistance of C. neoformans is imperative. The analysis of functional genomics data and genome-scale mutant libraries may facilitate the genetic dissection of such complex phenotypes but with limited efficiency. Here, we present a genome-scale co-functional network for C. neoformans, CryptoNet, which covers ~81% of the coding genome and provides an efficient intermediary between functional genomics data and reverse-genetics resources for the genetic dissection of C. neoformans phenotypes. CryptoNet is the first genome-scale co-functional network for any fungal pathogen. CryptoNet effectively identified novel genes for pathogenicity and drug resistance using guilt-by-association and context-associated hub algorithms. CryptoNet is also the first genome-scale co-functional network for fungi in the basidiomycota phylum, as Saccharomyces cerevisiae belongs to the ascomycota phylum. CryptoNet may therefore provide insights into pathway evolution between two distinct phyla of the fungal kingdom. The CryptoNet web server (www.inetbio.org/cryptonet) is a public resource that provides an interactive environment of network-assisted predictive genetics for C. neoformans.

Systematic functional profiling of transcription factor networks in Cryptococcus neoformans.

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.

Msi1-Like (MSIL) Proteins in Fungi.

Msi1-like (MSIL) proteins, which are eukaryote-specific and contain a series of WD40 repeats, have pleiotropic roles in chromatin assembly, DNA damage repair, and regulation of nutrient/stress-sensing signaling pathways. In the fungal kingdom, the functions of MSIL proteins have been studied most intensively in the budding yeast model Saccharomyces cerevisiae, an ascomycete. Yet their functions are largely unknown in other fungi. Recently, an MSIL protein, Msl1, was discovered and functionally characterized in the pathogenic yeast Cryptococcus neoformans, a basidiomycete. Interestingly, MSIL proteins appear to have redundant and unique roles in both fungi, suggesting that MSIL proteins may have evolutionarily divergent roles in different parts of the fungal kingdom. In this review, we will describe the current findings regarding the role of MSIL proteins in fungi and discuss future directions for research on this topic.

Functional Characterization of cAMP-Regulated Gene, CAR1, in Cryptococcus neoformans.

The cyclic AMP (cAMP) pathway plays a major role in growth, sexual differentiation, and virulence factor synthesis of pathogenic fungi. In Cryptococcus neoformans, perturbation of the cAMP pathway, such as a deletion in the gene encoding adenylyl cyclase (CAC1), causes defects in the production of virulence factors, including capsule and melanin production, as well as mating. Previously, we performed a comparative transcriptome analysis of the Ras- and cAMP-pathway mutants, which revealed 163 potential cAMP-regulated genes (38 genes at a 2-fold cutoff). The present study characterized the role of one of the cAMP pathway-dependent genes (serotype A identification number CNAG_ 06576.2). The expression patterns were confirmed by Northern blot analysis and the gene was designated cAMP-regulated gene 1 (CAR1). Interestingly, deletion of CAR1 did not affect biosynthesis of any virulence factors and the mating process, unlike the cAMP-signaling deficient cac1Δ mutant. Furthermore, the cac1Δ mutant exhibited wild-type levels of the stress-response phenotype against diverse environmental cues, indicating that Car1, albeit regulated by the cAMP-pathway, is not essential to confer a cAMP-dependent phenotype in C. neoformans.

Comparative transcriptome analysis of the CO2 sensing pathway via differential expression of carbonic anhydrase in Cryptococcus neoformans.

Carbon dioxide (CO(2)) sensing and metabolism via carbonic anhydrases (CAs) play pivotal roles in survival and proliferation of pathogenic fungi infecting human hosts from natural environments due to the drastic difference in CO(2) levels. In Cryptococcus neoformans, which causes fatal fungal meningoencephalitis, the Can2 CA plays essential roles during both cellular growth in air and sexual differentiation of the pathogen. However the signaling networks downstream of Can2 are largely unknown. To address this question, the present study employed comparative transcriptome DNA microarray analysis of a C. neoformans strain in which CAN2 expression is artificially controlled by the CTR4 (copper transporter) promoter. The P(CTR4)CAN2 strain showed growth defects in a CO(2)-dependent manner when CAN2 was repressed but resumed normal growth when CAN2 was overexpressed. The Can2-dependent genes identified by the transcriptome analysis include FAS1 (fatty acid synthase 1) and GPB1 (G-protein beta subunit), supporting the roles of Can2 in fatty acid biosynthesis and sexual differentiation. Cas3, a capsular structure designer protein, was also discovered to be Can2-dependent and yet was not involved in CO(2)-mediated capsule induction. Most notably, a majority of Can2-dependent genes were environmental stress-regulated (ESR) genes. Supporting this, the CAN2 overexpression strain was hypersensitive to oxidative and genotoxic stress as well as antifungal drugs, such as polyene and azole drugs, potentially due to defective membrane integrity. Finally, an oxidative stress-responsive Atf1 transcription factor was also found to be Can2-dependent. Atf1 not only plays an important role in diverse stress responses, including thermotolerance and antifungal drug resistance, but also represses melanin and capsule production in C. neoformans. In conclusion, this study provides insights into the comprehensive signaling networks orchestrated by CA/CO(2)-sensing pathways in pathogenic fungi.