Step-wise elimination of α-mitochondrial nucleoids and mitochondrial structure as a basis for the strict uniparental inheritance in Cryptococcus neoformans.

In most sexual eukaryotes, mitochondrial (mt) DNA is uniparentally inherited, although the detailed mechanisms underlying this phenomenon remain controversial. The most widely accepted explanations include the autophagic elimination of paternal mitochondria in the fertilized eggs and the active degradation of paternal mitochondrial DNA. To decode the precise program for the uniparental inheritance, we focused on Cryptococcus neoformans as a model system, in which mtDNA is inherited only from the a-parent, although gametes of a- and α-cells are of equal size and contribute equal amounts of mtDNA to the zygote. In this research, the process of preferential elimination of the mitochondria contributed by the α-parent (α-mitochondria) was studied by fluorescence microscopy and single cell analysis using optical tweezers, which revealed that α-mitochondria are preferentially reduced by the following three steps: (1) preferential reduction of α-mitochondrial (mt) nucleoids and α-mtDNA, (2) degradation of the α-mitochondrial structure and (3) proliferation of remaining mt nucleoids during the zygote development. Furthermore, AUTOPHAGY RELATED GENE (ATG) 8 and the gene encoding mitochondrial endonuclease G (NUC1) were disrupted, and the effects of their disruption on the uniparental inheritance were scrutinized. Disruption of ATG8 (ATG7) and NUC1 did not have severe effects on the uniparental inheritance, but microscopic examination revealed that α-mitochondria lacking mt nucleoids persisted in Δatg8 zygotes, indicating that autophagy is not critical for the uniparental inheritance per se but is responsible for the clearance of mitochondrial structures after the reduction of α-mt nucleoids.

[A Silkworm Infection Model to Evaluate Antifungal Drugs for Cryptococcosis].

The development of effective drugs against fungal diseases involves performing infection experiments in animals to evaluate candidate therapeutic compounds. Cryptococcus neoformans is a pathogenic fungus that causes deep mycosis, resulting in respiratory illness and meningitis. Here we describe a silkworm system established to evaluate the safety and efficacy of therapeutic drugs against infection by Cryptococcus neoformans and the advantages of this system over other animal models. The silkworm assay system has two major advantages: 1) silkworms are less expensive to rear and their use is less problematic than that of mammals in terms of animal welfare, and 2) in vivo screenings for identifying candidate drugs can be easily performed using a large number of silkworms. The pharmacokinetics of compounds are consistent between silkworms and mammals. Moreover, the ED50 values of antibiotics are concordant between mammalian and silkworm infection models. Furthermore, the body size of silkworms makes them easy to handle in experimental procedures compared with other invertebrate infectious experimental systems, and accurate amounts of pathogens and chemicals can be injected fairly easily. These advantages of silkworms as a host animal make them useful for screening candidate drugs for cryptococcosis.

Temperature during conidiation affects stress tolerance, pigmentation, and trypacidin accumulation in the conidia of the airborne pathogen Aspergillus fumigatus.

Asexual spores (conidia) are reproductive structures that play a crucial role in fungal distribution and survival. As fungal conidia are, in most cases, etiological agents of plant diseases and fungal lung disease, their stress resistance and interaction with their hosts have drawn increasing attention. In the present study, we investigated whether environmental temperature during conidiation affects the stress tolerance of the conidia of the human pathogenic fungus Aspergillus fumigatus. Conidia from a 25°C culture showed a lower tolerance to heat (60°C) and oxidative (H2O2) stresses and a marked resistance to ultraviolet radiation exposure, compared with those produced at 37 and 45°C. The accumulation of trehalose was lower in the conidia from the 25°C culture. Furthermore, the conidia from the 25°C culture showed darker pigmentation and increased transcripts of dihydroxynaphthalene (DHN)-melanin biosynthesis-related genes (i.e., pksP, arp1, and arp2). An RNA-sequencing analysis revealed that the transcription level of the trypacidin (tpc) gene cluster, which contains 13 genes, was sharply and coordinately activated in the conidia from the 25°C culture. Accordingly, trypacidin was abundant in the conidia from the 25°C culture, whereas there was little trypacidin in the conidia from the 37°C culture. Taken together, these data show that the environmental temperature during conidiation affects conidial properties such as stress tolerance, pigmentation, and mycotoxin accumulation. To enhance our knowledge, we further explored the temperature-dependent production of DHN-melanin and trypacidin in clinical A. fumigatus isolates. Some of the isolates showed temperature-independent production of DHN-melanin and/or trypacidin, indicating that the conidia-associated secondary metabolisms differed among the isolates.

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions.

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).

Putative orotate transporter of Cryptococcus neoformans, Oat1, is a member of the NCS1/PRT transporter super family and its loss causes attenuation of virulence.

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura+. We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.

Comparative transcriptome analysis revealing dormant conidia and germination associated genes in Aspergillus species: an essential role for AtfA in conidial dormancy.

BACKGROUND: Fungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae. RESULTS: We found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1 h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63 % of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy. CONCLUSIONS: A comparison among transcriptomes of hyphae, resting conidia, and 1 h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.

Identification of genes involved in the phosphate metabolism in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that can cause life-threatening meningoencephalitis in immuno-compromized patients. To propagate in the human body, this organism has to acquire phosphate that functions in cellular signaling pathways and is also an essential component of nucleic acids and phospholipids. Thus it is reasonable to assume that C. neoformans (Cn) possesses a phosphate regulatory system (PHO system) analogous to that of other fungi. By BLAST searches using the amino acid sequences of the components of the PHO system of Saccharomyces cerevisiae (Sc), we found potential counterparts to ScPHO genes in C. neoformans, namely, acid phosphatase (CnPHO2), the cyclin-dependent protein kinase (CDK) inhibitor (CnPHO81), Pho85-cyclin (CnPHO80), and CDK (CnPHO85). Disruption of each candidate gene, except CnPHO85, followed by phenotypic analysis, identified most of the basic components of the CnPHO system. We found that CnPHO85 was essential for the growth of C. neoformans, having regulatory function in the CnPHO system. Genetic screening and ChIP analysis, showed that CnPHO4 encodes a transcription factor that binds to the CnPHO genes in a Pi-dependent manner. By RNA-seq analysis of the wild-type and the regulatory mutants of the CnPHO system, we found C. neoformans genes whose expression is controlled by the regulators of the CnPHO system. Thus the CnPHO system shares many properties with the ScPHO system, but expression of those CnPHO genes that encode regulators is controlled by phosphate starvation, which is not the case in the ScPHO system (except ScPHO81). We also could identify some genes involved in the stress response of the pathogenic yeast, but CnPho4 appeared to be responsible only for phosphate starvation.

Effects of the F-actin inhibitor latrunculin A on the budding yeast Saccharomyces cerevisiae.

Our basic cell biology research was aimed at investigating the effect on eukaryotic cells of the sudden loss of the F-actin cytoskeleton. Cells treated with latrunculin A (LA) in yeast extract peptone dextrose (YEPD) medium were examined using phase-contrast and fluorescent microscopy, freeze-substitution, transmission and scanning electron microscopy, counted using a Bürker chamber and their absorbance measured. The cells responded to the presence of LA, an F-actin inhibitor, with the disappearance of actin patches, actin cables and actin rings. This resulted in the formation of larger spherical cells with irregular morphology in the cell walls and ultrastructural disorder of the cell organelles and secretory vesicles. Instead of buds, LA-inhibited cells formed only 'table-mountain-like' wide flattened swellings without apical growth with a thinner glucan cell-wall layer containing ß-1,3-glucan microfibrils. The LA-inhibited cells lysed. Actin cables and patches were required for bud formation and bud growth. In addition, actin patches were required for the formation of ß-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.

Genome sequence comparison of Aspergillus fumigatus strains isolated from patients with pulmonary aspergilloma and chronic necrotizing pulmonary aspergillosis.

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

Positional cloning in Cryptococcus neoformans and its application for identification and cloning of the gene encoding methylenetetrahydrofolate reductase.

Cryptococcus neoformans, a basidiomycetous human pathogenic yeast, has been widely used in research fields in medical mycology as well as basic biology. Gene cloning or identification of the gene responsible for a mutation of interest is a key step for functional analysis of a particular gene. The availability therefore, of the multiple methods for cloning is desirable. In this study, we proposed a method for a mapping-based gene identification/cloning (positional cloning) method in C. neoformans. To this end, we constructed a series of tester strains, one of whose chromosomes was labeled with the URA5 gene. A heterozygous diploid constructed by crossing one of the tester strains to a mutant strain of interest loses a chromosome(s) spontaneously, which is the basis for assigning a recessive mutant gene to a particular chromosome in the mitotic mapping method. Once the gene of interest is mapped to one of the 14 chromosomes, classical genetic crosses can then be performed to determine its more precise location. The positional information thus obtained can then be used to significantly narrow down candidate genes by referring to the Cryptococcus genome database. Each candidate gene is then examined whether it would complement the mutation. We successfully applied this method to identify CNA07390 encoding methylenetetrahydrofolate reductase as the gene responsible for a methionine-requiring mutant in our mutant collection.

Dendritic cell-based immunization ameliorates pulmonary infection with highly virulent Cryptococcus gattii.

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.

The role of AtfA and HOG MAPK pathway in stress tolerance in conidia of Aspergillus fumigatus.

Aspergillus fumigatus is a life-threatening pathogenic fungus, whose conidium is the infectious agent of aspergillosis. To better understand the mechanism underlying the long-term viability of conidia, we characterized a bZip transcription factor, AtfA, with special reference to stress-tolerance in conidia. The atfA deletion mutant conidia showed significant sensitivity to high temperature and oxidative stress. The trehalose content that accumulated in conidia was reduced in the mutant conidia. Transcriptome analysis revealed that AtfA regulated several stress-protection-related genes such as catA, dprA, scf1, and conJ at the conidiation stage. The upstream high-osmolarity glycerol pathway was also involved in conferring stress tolerance in conidia because ΔpbsB showed stress sensitivity and reduced trehalose in conidia. However, a mutant lacking the SakA mitogen-activated protein kinase (MAPK) produced normal conidia. We investigated another MAPK, MpkC, in relation with SakA, and the double deletion mutant, ΔsakA,mpkC, was defective in conidia stress tolerance. We concluded that MpkC is able to bypass SakA, and the two MAPKs redundantly regulate the conidia-related function of AtfA in A. fumigatus.

Whole-genome comparison of Aspergillus fumigatus strains serially isolated from patients with aspergillosis.

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.

Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans.

Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence.

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans.

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1-A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5-fluoroorotic acid treatment of ORF4-expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid-free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1-A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.

The CRZ1/SP1-like gene links survival under limited aeration, cell integrity and biofilm formation in the pathogenic yeast Cryptococcus neoformans.

AIMS: Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. METHODS: Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. RESULTS: We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. CONCLUSIONS: Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.

The effects of the F-actin inhibitor latrunculin A on the pathogenic yeast Cryptococcus neoformans.

BACKGROUND: This basic research aimed to investigate the effects of the actin inhibitor latrunculin A (LA) on the human pathogen Cryptococcus neoformans, by freeze-substitution (FS) and electron microscopy (EM), to determine whether the actin cytoskeleton can become a new antifungal target for inhibition of cell division. METHODS: Cells treated with LA for 20 h in yeast-extract peptone dextrose medium were investigated by phase-contrast and fluorescent microscopy, FS and transmission EM, counted in a Bürker chamber and the absorbance was then measured. RESULTS: The disappearance of actin patches, actin cables and actin rings demonstrated the response of the cells of C. neoformans to the presence of the actin inhibitor LA. The removal of actin cables and patches arrested proliferation and led to the production of cells that had ultrastructural disorder, irregular morphology of the mitochondria and thick aberrant cell walls. Budding cells lysed in the buds and septa. CONCLUSION: LA exerts fungistatic, fungicidal and fungilytic effects on the human pathogenic yeast C. neoformans.

NikA/TcsC histidine kinase is involved in conidiation, hyphal morphology, and responses to osmotic stress and antifungal chemicals in Aspergillus fumigatus.

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans.

UNLABELLED: Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE: Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.