Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Cryptococcus neoformans requires the TVF1 gene for thermotolerance and virulence.

Cryptococcus neoformans is the primary causative agent of cryptococcosis. Since C. neoformans thrives in environments and its optimal growth temperature is 25-30°C, it needs to adapt to heat stress in order to cause infection in mammalian hosts. In this study, we aimed to investigate the role of an uncharacterized gene, CNAG_03308. Although the CNAG_03308 deletion strain grew as well as the parent strain KN99, it produced yeast cells with abnormal morphology at 37°C and failed to propagate at 39°C. Furthermore, the deletion strain exhibited slower growth at 37°C in the presence of congo red, which is a cell wall stressor. When cultured at 39°C, the deletion strain showed strong staining with fluorescent probes for cell wall chitin and chitosan, including FITC-labeled wheat germ agglutinin, Eosin Y, and calcofluor white. The transmission electron microscopy of the deletion strain revealed a thickened inner layer of the cell wall containing chitin and chitosan under heat stress. This cell-surface altered deletion strain induced dendritic cells to secrete more interleukin (IL)-6 and IL-23 than the control strains under heat stress. In a murine infection study, C57BL/6 mice infected with the deletion strain exhibited lower mortality and lower fungal burden in the lungs and brain compared to those infected with the control strains. Based on these findings, we concluded that CNAG_03308 gene is necessary for C. neoformans to adapt to heat stress both in vitro and in the host environment. Therefore, we designated the CNAG_03308 gene as TVF1, which stands for thermotolerance and virulence-related factor 1.

Cryptococcus neoformans is a fungal pathogen causing cryptococcosis, which requires thermotolerance to proliferate in the host environment. In the present study, we identified a novel gene, TVF1 (CNAG_03308), required for thermotolerance and virulence by reverse genetics approach.

Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species.

Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

MDA5 signaling induces type 1 IFN- and IL-1-dependent lung vascular permeability which protects mice from opportunistic fungal infection.

Lungs balance threat from primary viral infection, secondary infection, and inflammatory damage. Severe pulmonary inflammation induces vascular permeability, edema, and organ dysfunction. We previously demonstrated that poly(I:C) (pICLC) induced type 1 interferon (t1IFN) protected mice from Cryptococcus gattii (Cg) via local iron restriction. Here we show pICLC increased serum protein and intravenously injected FITC-dextran in the lung airspace suggesting pICLC induces vascular permeability. Interestingly, pICLC induced a pro-inflammatory signature with significant expression of IL-1 and IL-6 which depended on MDA5 and t1IFN. Vascular permeability depended on MDA5, t1IFN, IL-1, and IL-6. T1IFN also induced MDA5 and other MDA5 signaling components suggesting that positive feedback contributes to t1IFN dependent expression of the pro-inflammatory signature. Vascular permeability, induced by pICLC or another compound, inhibited Cg by limiting iron. These data suggest that pICLC induces t1IFN which potentiates pICLC-MDA5 signaling increasing IL-1 and IL-6 resulting in leakage of antimicrobial serum factors into lung airspace. Thus, induced vascular permeability may act as an innate defense mechanism against opportunistic fungal infection, such as cryptococcosis, and may be exploited as a host-directed therapeutic target.

Novel angular naphthopyrone formation by Arp1p dehydratase involved in Aspergillus fumigatus melanin biosynthesis.

Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8-dihydroxynaphthalene (DHN)-melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN-melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8-THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non-enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 µM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish-green conidial pigmentation of A. fumigatus.

Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection.

The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.

Moderate levels of 5-fluorocytosine cause the emergence of high frequency resistance in cryptococci.

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.

Antifungal Susceptibility Profiles of Olorofim (Formerly F901318) and Currently Available Systemic Antifungals against Mold and Yeast Phases of Talaromyces marneffei.

In vitro antifungal susceptibility profiling of 32 clinical and environmental Talaromyces marneffei isolates recovered from southern China was performed against olorofim and 7 other systemic antifungals, including amphotericin B, 5-flucytosine, posaconazole, voriconazole, caspofungin, and terbinafine, using CLSI methodology. In comparison, olorofim was the most active antifungal agent against both mold and yeast phases of all tested Talaromyces marneffei isolates, exhibiting an MIC range, MIC50, and MIC90 of 0.0005 to 0.002 µg/ml, 0.0005 µg/ml, and 0.0005 µg/ml, respectively.

A New Lineage of Cryptococcus gattii (VGV) Discovered in the Central Zambezian Miombo Woodlands.

We discovered a new lineage of the globally important fungal pathogen Cryptococcus gattii on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of C. gattii revealed a unique suite of genes together with gene loss and inversion events. However, standard URA5 restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new URA5 RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens.IMPORTANCECryptococcus gattii is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that C. gattii falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety gattii five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause.

A Novel Role of Fungal Type I Myosin in Regulating Membrane Properties and Its Association with d-Amino Acid Utilization in Cryptococcus gattii.

We found a novel role of Myo5, a type I myosin (myosin-I), and its fortuitous association with d-amino acid utilization in Cryptococcus gattii Myo5 colocalized with actin cortical patches and was required for endocytosis. Interestingly, the myo5Δ mutant accumulated high levels of d-proline and d-alanine which caused toxicity in C. gattii cells. The myo5Δ mutant also accumulated a large set of substrates, such as membrane-permeant as well as non-membrane-permeant dyes, l-proline, l-alanine, and flucytosine intracellularly. Furthermore, the efflux rate of fluorescein was significantly increased in the myo5Δ mutant. Importantly, the endocytic defect of the myo5Δ mutant did not affect the localization of the proline permease and flucytosine transporter. These data indicate that the substrate accumulation phenotype is not solely due to a defect in endocytosis, but the membrane properties may have been altered in the myo5Δ mutant. Consistent with this, the sterol staining pattern of the myo5Δ mutant was different from that of the wild type, and the mutant was hypersensitive to amphotericin B. It appears that the changes in sterol distribution may have caused altered membrane permeability in the myo5Δ mutant, allowing increased accumulation of substrate. Moreover, myosin-I mutants generated in several other yeast species displayed a similar substrate accumulation phenotype. Thus, fungal type I myosin appears to play an important role in regulating membrane permeability. Although the substrate accumulation phenotype was detected in strains with mutations in the genes involved in actin nucleation, the phenotype was not shared in all endocytic mutants, indicating a complicated relationship between substrate accumulation and endocytosis.IMPORTANCECryptococcus gattii, one of the etiological agents of cryptococcosis, can be distinguished from its sister species Cryptococcus neoformans by growth on d-amino acids. C. gattiiMYO5 affected the growth of C. gattii on d-amino acids. The myo5Δ cells accumulated high levels of various substrates from outside the cells, and excessively accumulated d-amino acids appeared to have caused toxicity in the myo5Δ cells. We provide evidence on the alteration of membrane properties in the myo5Δ mutants. Additionally, alteration in the myo5Δ membrane permeability causing higher substrate accumulation is associated with the changes in the sterol distribution. Furthermore, myosin-I in three other yeasts also manifested a similar role in substrate accumulation. Thus, while fungal myosin-I may function as a classical myosin-I, it has hitherto unknown additional roles in regulating membrane permeability. Since deletion of fungal myosin-I causes significantly elevated susceptibility to multiple antifungal drugs, it could serve as an effective target for augmentation of fungal therapy.

Pulmonary Iron Limitation Induced by Exogenous Type I IFN Protects Mice from Cryptococcus gattii Independently of T Cells.

Cryptococcus neoformans causes deadly mycosis primarily in AIDS patients, whereas Cryptococcus gattii infects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence of C. gattii As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome of C. neoformans and C. gattii infections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. In C. neoformans-infected mice, pICLC activity was associated with C. neoformans containment and classical Th1 immunity. In contrast, pICLC activity against C. gattii did not require any immune factors previously associated with C. neoformans immunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly, C. gattii pICLC activity depended on ß-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggesting C. gattii pICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcus in vitro, suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection by C. gattii and C. neoformans but by distinct mechanisms; the C. gattii effect was mediated by iron limitation, while the effect on C. neoformans infection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for different Cryptococcus species suggests a possible mechanism by which HIV infection may select against C. gattii but not C. neoformansIMPORTANCECryptococcus neoformans and Cryptococcus gattii cause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species, C. gattii infects very few AIDS patients, while C. neoformans infection is an AIDS-defining illness, suggesting that the host response to HIV selects C. neoformans over C. gattii We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action against C. neoformans was due to activation of well-defined immune pathways known to deter C. neoformans, whereas these immune pathways were dispensable for pICLC treatment of C. gattii Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to control C. neoformans infection but is protective against C. gattii Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibited C. gattii, thus suggesting an entirely new mode of nutritional immunity activated by viral signals.

Cryptococcus neoformans, Unlike Candida albicans, Forms Aneuploid Clones Directly from Uninucleated Cells under Fluconazole Stress.

Heteroresistance to fluconazole (FLC) in Cryptococcus is a transient adaptive resistance which is lost upon release from the drug pressure. It is known that clones heteroresistant to FLC invariably contain disomic chromosomes, but how disomy is formed remains unclear. Previous reports suggested that the aneuploid heteroresistant colonies in Cryptococcus emerge from multinucleated cells, resembling the case in Candida albicans Although a small number of cells containing multiple nuclei appear in a short time after FLC treatment, we provide evidence that the heteroresistant colonies in the presence of FLC arise from uninucleate cells without involving multinuclear/multimeric stages. We found that fidelity of chromosome segregation in mitosis plays an important role in regulation of FLC heteroresistance frequency in C. neoformans Although FLC-resistant colonies occurred at a very low frequency, we were able to modulate the frequency of heteroresistance by overexpressing SMC1, which encodes a protein containing an SMC domain in chromosome segregation. Using time-lapse microscopy, we captured the entire process of colony formation from a single cell in the presence of FLC. All the multinucleated cells formed within a few hours of FLC exposure failed to multiply after a few cell divisions, and the cells able to proliferate to form colonies were all uninucleate without exception. Furthermore, no nuclear fusion event or asymmetric survival between mother and daughter cells, a hallmark of chromosome nondisjunction in haploid organisms, was observed. Therefore, the mechanisms of aneuploidy formation in C. neoformans appear different from most common categories of aneuploid formation known for yeasts.IMPORTANCE The gold standard of cryptococcosis treatment consists of induction therapy with amphotericin B followed by lifelong maintenance therapy with fluconazole (FLC). However, prolonged exposure to FLC induces the emergence of clones heteroresistant to azoles. All the heteroresistant clones thus far analyzed have been shown to be aneuploids, but how the aneuploid is formed remains unclear. Aneuploidy in fungi and other eukaryotic cells is known to result most commonly from chromosome missegregation during cell division due to defects in any one of the multiple components and processes that are required for the formation of two genetically identical daughter cells. Although formation of multinucleated cells has been observed in cells exposed to FLC, evidence for the emergence of drug-resistant aneuploid populations directly from such cells has been lacking. We show the evidence that the aneuploid in fluconazole-heteroresistant clones of Cryptococcus neoformans is derived neither from multinucleated cells nor from chromosome missegregation.

Exogenous Stimulation of Type I Interferon Protects Mice with Chronic Granulomatous Disease from Aspergillosis through Early Recruitment of Host-Protective Neutrophils into the Lung.

Invasive aspergillosis (IA) remains the primary cause of morbidity and mortality in chronic granulomatous disease (CGD) patients, often due to infection by Aspergillus species refractory to antifungals. This motivates the search for alternative treatments, including immunotherapy. We investigated the effect of exogenous type I interferon (IFN) activation on the outcome of IA caused by three Aspergillus species, A. fumigatus, A. nidulans, and A. tanneri, in CGD mice. The animals were treated with poly(I):poly(C) carboxymethyl cellulose poly-l-lysine (PICLC), a mimetic of double-stranded RNA, 24 h preinfection and postinfection. The survival rates and lung fungal burdens were markedly improved by PICLC immunotherapy in animals infected with any one of the three Aspergillus species. While protection from IA was remarkable, PICLC induction of type I IFN in the lungs surged 24 h posttreatment and returned to baseline levels by 48 h, suggesting that PICLC altered early events in protection against IA. Immunophenotyping of recruited leukocytes and histopathological examination of tissue sections showed that PICLC induced similar cellular infiltrates as those in untreated-infected mice, in both cases dominated by monocytic cells and neutrophils. However, the PICLC immunotherapy resulted in a marked earlier recruitment of the leukocytes. Unlike with conidia, infection with A. nidulans germlings reduced the protective effect of PICLC immunotherapy. Additionally, antibody depletion of neutrophils totally reversed the protection, suggesting that neutrophils are crucial for PICLC-mediated protection. Together, these data show that prophylactic PICLC immunotherapy prerecruits these cells, enabling them to attack the conidia and thus resulting in a profound protection from IA.IMPORTANCE Patients with chronic granulomatous disease (CGD) are highly susceptible to invasive aspergillosis (IA). While Aspergillus fumigatus is the most-studied Aspergillus species, CGD patients often suffer IA caused by A. nidulans, A. tanneri, and other rare species. These non-fumigatus Aspergillus species are more resistant to antifungal drugs and cause higher fatality rates than A. fumigatus Therefore, alternative therapies are needed to protect CGD patients. We report an effective immunotherapy of mice infected with three Aspergillus species via PICLC dosing. While protection from IA was long lasting, PICLC induction of type I IFN surged but quickly returned to baseline levels, suggesting that PICLC was altering early events in IA. Interestingly, we found responding immune cells to be similar between PICLC-treated and untreated-infected mice. However, PICLC immunotherapy resulted in an earlier recruitment of the leukocytes and suppressed fungal growth. This study highlights the value of type I IFN induction in CGD patients.

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

The Case for Adopting the "Species Complex" Nomenclature for the Etiologic Agents of Cryptococcosis.

Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using "Cryptococcus neoformans species complex" and "C. gattii species complex" as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.

The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence.

Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus.

Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis.

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

Differences between Cryptococcus neoformans and Cryptococcus gattii in the Molecular Mechanisms Governing Utilization of D-Amino Acids as the Sole Nitrogen Source.

The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate Cryptococcus gattii from C. neoformans. We used the C. neoformans H99 and C. gattii R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (DAO) genes in both strains and showed that each DAO gene plays different roles in D-amino acid utilization in each strain. Deletion of DAO2 retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 DAO genes contributed to growth on D-Asn and D-Asp. DAO3 was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of DAO1 or DAO3 further exacerbated it on four D-amino acids. Overexpression of DAO2 or DAO3 enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native DAO genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the DAO2 genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all DAO genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the DAO genes in R265. These results suggest that C. neoformans and C. gattii divergently evolved in D-amino acid utilization influenced by their major ecological niches.

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Molecular mechanisms of hypoxic responses via unique roles of Ras1, Cdc24 and Ptp3 in a human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.