Hannaella oleicumulans sp. nov. and Hannaella higashiohmiensis sp. nov., two novel oleaginous basidiomycetous yeast species.

Three strains of novel oleaginous yeast species were isolated from soil samples collected in Shiga Prefecture, Japan. The sequences of the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit (LSU) of the rRNA genes indicated that these novel yeast species are members of the genus Hannaella. The results of molecular phylogenetic analysis indicated that strains 38-3 and 8s1 were closely related to Hannaella oryzae. They differed by 10 nucleotide substitutions and one gap (1.77 %) in the D1/D2 region of the LSU of the rRNA genes and by 17-18 nucleotide substitutions and 10-11 gaps (5.45-5.85 %) in the ITS region. Strain 51-4 differed from the type strain of the most closely related species, Hannaella pagnoccae, by 26 nucleotide substitutions (4.46 %) in the D1/D2 region of the LSU of the rRNA genes and by 20 nucleotide substitutions and six gaps (5.42 %) in the ITS region. The names proposed for these previously undescribed species are Hannaella oleicumulans sp. nov. and Hannaella higashiohmiensis sp. nov.

Emergence of [GAR+ ] cells in yeast from sake brewing affects the fermentation properties.

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR+ ] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR+ ] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR+ ] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR+ ] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR+ ] cells in the industrial sake strains but not in the kimoto strains.

Glazing Affects the Fermentation Process of Sake Brewed in Pottery.

Sake (Japanese rice wine) was fermented in pottery for more than a millennium before wooden barrels were adopted to obtain a greater brewing capacity. Although a recently conducted analysis of sake brewed in pottery indicated that sake brewed in unglazed pottery contains more ethanol than that brewed in glazed pottery, little is known about the characteristics of sake brewed in pottery. In this study, we used two types of ceramic containers of identical size, one glazed and one unglazed, for small-scale sake brewing to evaluate the effects of glazing on fermentation properties. The following parameters were measured continuously in the sake samples over 3 weeks of fermentation: temperature, weight, ethanol concentration, and glucose concentration in sake mash. Taste-sensory values, minerals, and volatile components were also quantified in the final fermented sake mash. The results show that, in the unglazed containers, the temperature of the sake mash was lower and the weight loss was higher compared to the sake mash in the glazed containers. The quantity of ethanol and the levels of Na+, Fe3+, and Al3+ tended to be higher in the sake brewed in the unglazed pottery. A taste-sensory analysis revealed that umami and saltiness were also higher in the samples brewed in the unglazed pottery. These results suggest that glazing affects multiple fermentation parameters and the flavor of sake brewed in pottery. They may also suggest that the materials of the containers used in sake brewing generally affect the fermentation properties.

Lentilactobacillus buchneri domination during the fermentation of Japanese traditional fermented fish (funazushi).

Funazushi is a Japanese traditional fermented fish made with boiled rice without the addition of microbial starter cultures. Isolates from various commercial funazushi products, as identified by 16S rDNA sequences, suggested that Lentilactobacillus buchneri strains are major lactic acid bacteria. Based on an analysis of the putative CRISPR (clustered regularly interspaced short palindromic repeat) region, the genetic diversity of L. buchneri strains was examined. The data suggested that the diversity of L. buchneri strains depended on the factories at which funazushi was produced. An analysis of samples during fermentation indicated that the transition of microbes occurred, and L. buchneri was the dominant species. To determine the factors associated with domination, bacteriocin production and environmental stress tolerance, including NaCl and organic acid (lactate and acetate) tolerance, were evaluated. L. buchneri isolates did not produce bacteriocin. Although the isolates did not exhibit NaCl tolerance, they displayed higher lactate tolerance than other lactic acid bacteria isolated during funazushi fermentation. Based on reports that L. buchneri can convert lactate to acetate, the previous and present results suggested that lactate tolerance and lactate conversion in L. buchneri could explain its domination in funazushi. Our study presented a model for the domination mechanisms of specific microbes in fermented foods by spontaneous fermentation.

Exhibition of antifungal resistance by sterol-auxotrophic strains of Candida glabrata with intact virulence.

Background: Candida glabrata is an emerging fungal pathogen in immune-compromised hosts. Previously undetected C. glabrata isolates were successfully recovered from clinical specimens by adding sterols to the growth medium. The clinical isolates are unable to synthesize ergosterol but can take up exogenous sterols under aerobic conditions. Objectives: This study characterizes the sterol-auxotrophic C. glabrata strains, examines the mutation(s) in sterol synthesis genes, characterizes the drug susceptibility and evaluates the virulence in a mouse infection model. Methods: Drug susceptibility of the C. glabrata strains was evaluated in a sterol-supplemented medium. The coding sequences of the sterol synthesis genes were analysed in six sterol-auxotrophic strains of C. glabrata. The fungal burden of mice infected with C. glabrata strain was determined. Results: The sterol-auxotrophic strains showed high-level resistance to both azoles and amphotericin B when sterols were supplied in the test medium. Additionally, the strains harbour missense mutations in either ERG1 or ERG7. Significant differences in fungal burden were not observed between the sterol-auxotrophic strain and the sterol-competent strain with the mice infection models. Conclusions: The sterol-auxotrophic C. glabrata strain investigated in this study seemed to maintain intact virulence, probably due to the supply of exogenous sterols from host organ(s). This suggests that exogenous sterol uptake develops antifungal resistance during infection.

Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant "hot spot 1" mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

Monitoring of Iron Depletion-Induced Mitophagy in Pathogenic Yeast.

Mitophagy, which is the degradation of mitochondria via selective autophagic machinery, is thought to be involved in regulating the mass and function of mitochondria. Methods for detection of mitophagy have been reported for several fungal cells including some budding yeast, methylotrophic yeast, and filamentous fungi. Mitophagy in Saccharomyces cerevisiae is activated under nitrogen-poor conditions; however, the regulatory mechanism of mitophagy in most fungi has not been elucidated. Here we describe methods to monitor mitophagy in the pathogenic yeast Candida glabrata under iron-depleted conditions but not under nitrogen starvation. This observation may provide some clues to elucidate the physiological roles of mitophagy in eukaryotes.

Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.

Localized Cutaneous Cryptococcosis: Summary of Reported Cases in Japan.

A 68-year-old male plasterer with no history of trauma presented to our clinic in March 2012 with a 16×14-mm ulcer that developed following a crushed small papule on the right anterior chest. In April 2012, the patient was referred to another hospital, where cutaneous cryptococcosis was diagnosed based on discharge culture results. The patient was treated with oral itraconazole at a dose of 150 mg/day for 10 weeks; however, the ulcer remained unchanged and he discontinued the treatment. In May 2014, when he revisited our clinic, the ulcer with crust had grown to 29×13 mm. No regional lymph node swelling was noted. India ink staining showed a yeast-like fungus with a thick, clear capsule. A cream-colored, viscous yeast-like colony was observed on Sabouraud dextrose agar. Genetic testing identified the isolate as Cryptococcus neoformans serotype A. The patient was negative for serum cryptococcal antigen. Neither chest radiography nor computed tomography revealed any abnormalities. The patient had no underlying disease. Oral fluconazole (400 mg/day for 12 weeks) was prescribed, resulting in scar formation. The patient has remained free of relapse for one year to date, since the end of treatment. Localized cutaneous cryptococcosis is not a commonly used disease name overseas. However, 36 cases of this disease have been reported in Japan (since in 1968). We herein report a new case with localized cutaneous cryptococcosis and summarize previously reported cases in Japan.

Iron-depletion promotes mitophagy to maintain mitochondrial integrity in pathogenic yeast Candida glabrata.

Candida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis.

[Disseminated Cryptococcosis with Eosinophilia and Elevated IgE in a Non-HIV-infected Patient].

In general, disseminated cryptococcosis usually occurs among immunocompromised patients, especially those with cell-mediated immunodeficiency, such as HIV-infected patients. We present herein a rare case of an apparently immunocompetent 33-year-old woman who developed disseminated cryptococcal diseases, which included meningitis and pneumonia with eosinophilia, and pulmonary tuberculosis during her disease course. Pneumonia with a diffuse micronodular pattern, immediately followed by meningitis, was diagnosed as disseminated cryptococcosis, because of the presence of yeast-like-fungi demonstrated by transbronchial lung biopsy and a positive cerebrospinal fluid (CSF) culture. In addition, the pneumonia exhibited eosinophilia in the peripheral blood and bronchoalveolar lavage fluid. Re-exacerbation of the pneumonia occurred approximately 3 weeks after onset, along with a sputum culture positive for Mycobacterium tuberculosis. Administration of anti-tuberculosis drugs resulted in recovery from the pulmonary tuberculosis. The treatment of cryptococcal meningitis was initiated using a standard induction regimen;however, an unrecovered status, highlighted by elevated CSF pressure, persisted. Finally, full recovery was induced by the addition of flucytosine treatment (100 mg/kg/day) and repeated daily via lumbar puncture. The allergic condition of this patient may have contributed to the onset of disseminated cryptococcosis.

Interferon-γ promotes phagocytosis of Cryptococcus neoformans but not Cryptococcus gattii by murine macrophages.

Among invasive fungal infections, cryptococcosis caused by inhalation of Cryptococcus neoformans or Cryptococcus gattii is particularly dangerous because it can disseminate to the central nervous system and cause life-threatening meningitis or meningoencephalitis. Previous reports described significant differences in the histopathological features of C. neoformans and C. gattii infection, such as greater pathogen proliferation and a limited macrophage response in mouse lung infected by C. gattii. To elucidate the difference in pathogenicity of these two Cryptococcus species, we investigated the interaction of C. neoformans and C. gattii with murine macrophages, the first line of host defense, by confocal laser microscopy. Only thin-capsulated, and not thick-capsulated C. neoformans and C. gattii were phagocytosed by macrophages. Preactivation with interferon-γ increased the phagocytic rate of thin-capsulated C. neoformans up to two-fold, but did not promote phagocytosis of thin-capsulated C. gattii. Lipopolysaccharide preactivation or Aspergillus fumigatus conidia co-incubation had no effect on internalization of thin-capsulated C. neoformans or C. gattii by macrophages. Phagocytosis of live thin-capsulated C. neoformans, but not that of live thin-capsulated C. gattii, induced interleukin-12 release from macrophages. However, phagocytosis of heat-killed or paraformaldehyde-fixed thin-capsulated C. neoformans did not increase IL-12 release, showing that the internalization of live yeast is important for initiating the immune response during C. neoformans-macrophage interactions. Our data suggest that macrophage response to C. gattii is limited compared with that to C. neoformans and that these results may partially explain the limited immune response and the greater pathogenicity of C. gattii.

The mannoprotein TIR3 (CAGL0C03872g) is required for sterol uptake in Candida glabrata.

Sterol uptake in the pathogenic fungus, Candida glabrata, occurs via the sterol transporter, CgAus1p. Azole inhibition of sterol biosynthesis can under certain circumstances be reversed by adding exogenously sterol. Here we demonstrate that the CgTIR3 (CAGL0C03872g) gene product is also required for sterol uptake, since Cgtir3Δ strains fail to take up sterol both aerobically and under hypoxic conditions. Western analysis using an HA-tagged TIR3 strain showed that CgTir3p localizes to the cell wall, and its expression is induced by serum. Semi-quantitative reverse transcriptase-PCR also showed that two transcription regulatory genes, CgUPC2A and CgUPC2B, control CgTIR3 as well as CgAUS1 gene expression. Interestingly, complementation studies using Cgtir3Δ showed that ScDAN1, a mannoprotein required for sterol uptake in Saccharomyces cerevisiae, could not complement the C. glabrata TIR3 function. Furthermore, sterol analyses, in which both the CgAUS1 and CgTIR3 genes were constitutively expressed, resulted in aerobic sterol uptake although the amount of uptake was considerably less than that of cells cultured aerobically with serum. These results suggest that additional factors other than CgAUS1 and CgTIR3 are required for sterol uptake in C. glabrata.

Potent drugs that attenuate anti-Candida albicans activity of fluconazole and their possible mechanisms of action.

Fluconazole (FLCZ) is a first-line drug for treating Candida albicans infections, but clinical failure due to reduced sensitivity is a growing concern. Our previous study suggested that certain drug combinations pose a particular challenge in potently reducing FLCZ's anti-C. albicans activity, and cyclooxygenase inhibitors formed the major group of these attenuating drugs in combination with FLCZ. In this study, we examined the effects of diclofenac sodium (DFNa) and related compounds in combination with FLCZ against C. albicans, and investigated their possible mechanisms of interaction. DFNa, ibuprofen, and omeprazole elevated the minimum inhibitory concentration (MIC) of FLCZ by 8-, 4-, and 4-fold, respectively; however, loxoprofen sodium and celecoxib did not. An analogue of DFNa, 2,6-dichlorodiphenylamine, also elevated the MIC by 4-fold. Gene expression analysis revealed that diclofenac sodium induced CDR1 efflux pump activity, but not CDR2 activity. In addition, an efflux pump CDR1 mutant, which was manipulated to not be induced by DFNa, showed less elevation of MIC compared to that shown by the wild type. Therefore, DFNa and related compounds are potent factors for reducing the sensitivity of C. albicans to FLCZ partly via induction of an efflux pump. Although it is not known whether such antagonism is relevant to the clinical treatment failure observed, further investigation of the molecular mechanisms underlying the reduction of FLCZ's anti-C. albicans activity is expected to promote safer and more effective use of the drug.

Breakthrough invasive Candida glabrata in patients on micafungin: a novel FKS gene conversion correlated with sequential elevation of MIC.

Candida glabrata strains sequentially isolated from blood developed resistance to micafungin (MICs from <0.015 to 4 µg/ml). A novel mutation identified in micafungin-resistant strains at bp 262 of FKS2 (containing a deletion of F659 [F659del]) was inserted into the homologous region in FKS1.

Genome-wide survey of transcriptional initiation in the pathogenic fungus, Candida glabrata.

DNA sequencing of the 5'-flanking region of the transcriptome effectively identifies transcription initiation sites and also aids in identifying unknown genes. This study describes a comprehensive polling of transcription start sites and an analysis of full-length complementary DNAs derived from the genome of the pathogenic fungus Candida glabrata. A comparison of the sequence reads derived from a cDNA library prepared from cells grown under different culture conditions against the reference genomic sequence of the Candida Genome Database (CGD: http://www.candidagenome.org/) revealed the expression of 4316 genes and their acknowledged transcription start sites (TSSs). In addition this analysis also predicted 59 new genes including 22 that showed no homology to the genome of Saccharomyces cerevisiae, a genetically close relative of C. glabrata. Furthermore, comparison of the 5'-untranslated regions (5'-UTRs) and core promoters of C. glabrata to those of S. cerevisiae showed various global similarities and differences among orthologous genes. Thus, the C. glabrata transcriptome can complement the annotation of the genome database and should provide new insights into the organization, regulation, and function of genes of this important human pathogen.

Detection of environmental sources of Histoplasma capsulatum in Chiang Mai, Thailand, by nested PCR.

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.

Histopathological study of murine pulmonary cryptococcosis induced by Cryptococcus gattii and Cryptococcus neoformans.

Although Cryptococcus gattii can cause life-threatening complications, putative virulence factors of C. gattii remain controversial. Therefore, we conducted the present study to elucidate the virulence factors of the yeast and found that the mortality rate of mice infected with C. gattii R265 was significantly higher than that of those infected with C. gattii 5815; however, no difference was found in the mortality rates between mice infected with C. gattii R265 and Cryptococcus neoformans H99. In contrast, we found a significant difference in histopathological findings of the lungs between mice infected with C. gattii R265 and C. neoformans H99. The former showed alveolar expansion due to yeast proliferation with much lesser macrophage response, whereas the latter showed numerous nodules in the alveolar space consisting of macrophages and multinucleated giant cells. Furthermore, alveolar expansion was more enhanced in mice infected with C. gattii R265 than in those infected with C. gattii 5815. Our study confirmed that there is a different pathophysiology leading to death during C. gattii and C. neoformans infections. The result can provide two characteristics of C. gattii: one includes some mechanisms to escape from host recognition via macrophage and another includes a high performance of pulmonary structural alteration. These characteristics may be associated with the high virulence of C. gattii.