Yeast Form Research Articles

Development of a novel β-1,6-glucan–specific detection system using functionally-modified recombinant endo-β-1,6-glucanase

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

An Unusual Case of Isolated Cryptococcal Osteomyelitis of Mastoid: An Enigma!

AbstractCryptococcosis is a disseminated fungal infection commonly affecting the lungs and the central nervous system in immunocompromised patients. Herein we report a rare case of isolated mastoid osteomyelitis due to Cryptococcus neoformans which was initially not recognized due to its uncommon presentation akin to a Betzold’s abscess. A 61-year-old male presented with complaints of left ear discharge for a 1-month duration associated with a swelling in the left side of the neck for which he underwent incision and drainage without any significant improvement. a contrast-enhanced computed tomography (CT) scan revealed extensive erosion of the left mastoid cavity with a collection. Intraoperatively, red friable granulation tissue was seen within the antrum, histopathology of which revealed capsulated yeast forms. The patient underwent left canal wall down mastoidectomy along with antifungal treatment. Bone involvement is rare in patients with cryptococcal infection with vertebrae being the most common site of cryptococcal osteomyelitis. The clinical presentation is nonspecific and can pose a diagnostic dilemma, as the condition can mimic both Betzold’s abscess as well as malignancy. CT findings and the use of specific fungal stains in histopathology will aid in diagnosing this condition. The purpose of this case report is to establish the first case of isolated cryptococcal mastoid osteomyelitis in the database of fungal osteomyelitis. The confirmation of fungal osteomyelitis should be based on histopathological examination. The possibility of fungal osteomyelitis should be borne in mind in any case of insidiously increasing mass with unclear etiology so that prompt antifungal therapy with surgical debridement is initiated.

Dendritic Cells Promote Treg Expansion but Not Th17 Generation in Response to Talaromyces marneffei Yeast Cells.

BackgroundDendritic cells (DCs) with both proinflammatory and tolerogenic properties have been implicated in modulation of CD4+ T cell responses in many fungal diseases. However, the role of DC in the context of Talaromyces marneffei (T. marneffei) infection has not been determined. In this study, we aimed to study the effect of the yeast form of T. marneffei yeasts on DCs, as well as the role of DCs in modulating T helper 17 (Th17) and regulatory T (Treg) cell responses to the pathogen.MethodsMouse bone marrow-derived DCs were stimulated with T. marneffei yeasts for 24 h. Frequencies of CD80 and CD86 expression on DCs and the levels of IL-6, IL-10 and TGF-β in the culture supernatant of yeast-stimulated DCs were detected by flow cytometry and ELISA, respectively. In co-culture experiments, CD4+ T lymphocytes of mice were isolated from the spleen using magnetic beads and co-cultured with T. marneffei yeasts, with or without DCs for 24 h. The proportions of Th17 and Treg cells in co-culture were detected by flow cytometry. The mRNA levels of RORγt and Foxp3 were detected by RT-PCR. Levels of IL-10 and TGF-β in the co-culture supernatant were detected by ELISA.ResultsThe expressions of CD80 and CD86 on DCs were increased, as well as IL-6, IL-10 and TGF-β levels in the culture supernatant of T. marneffei-stimulated DCs were higher than those in DCs cultured without T. marneffei. In co-culture experiments, in the presence of DCs, T. marneffei promoted Treg expansion and Foxp3 up-regulation but limited Th17 and downregulated RORγt. Levels of IL-10 and TGF-β were higher in the co-culture containing DCs than without DCs.ConclusionOur findings demonstrated that the interaction between DCs and T. marneffei could promote Treg expansion but not Th17 generation. These findings provide a mechanism by which DCs may promote immune tolerance in T. marneffei infection.

Endemic Fungi in Transplant and Immunocompromised Hosts: Epidemiology, Diagnosis, Treatment, and Prevention

Dimorphic endemic fungi exist as mold and yeast forms at room and body temperature within the host, respectively. Primary infection can arise from new environmental exposure and less frequently through organ transplantation. Reactivation of latent infection can occur when immunosuppression is introduced or intensified. Transplant and immunocompromised hosts (ICH) represent important populations at risk for complicated and disseminated forms of endemic mycoses (EM). Diagnosis is challenging as immunosuppression contributes to insidious clinical presentations resulting in diagnostic and treatment delays. Herein, we present updated diagnosis and prevention strategies for EM in the potential organ donor, transplant candidate and recipient, and ICH. Several EM have previously been considered as restricted to a limited geographical area; however, factors such as global warming, changing precipitation patterns, and increased travel are actively expanding the geographical distribution of endemic fungi. Serological testing, often used in the immunocompetent host, has significant limitations in transplant recipients and ICH. Fungal molecular methods have emerged as an important complementary diagnostic strategy for EM but would benefit from greater standardization and wider availability. High clinical suspicion and a multipronged diagnostic approach are recommended for timely diagnosis and prevention of complications. In ICH, combining diagnostic methods increases diagnostic yield for EM. Immunosuppression-specific risk assessment, laboratory monitoring, and targeted antifungal prophylaxis are pivotal to reduce EM burden. The use of molecular methods for early diagnosis and the role of newer azoles and immunomodulation in the management of EM require further investigation.

Laboratory Maintenance and Growth of Talaromyces marneffei.

Talaromyces marneffei is an important opportunistic human pathogen endemic to Southeast Asia. It is one of a number of pathogenic fungi that exhibits thermally controlled dimorphism. At 25°C, T. marneffei grows in a multicellular, filamentous hyphal form that can differentiate to produce dormant spores called conidia. These conidia are the likely infectious agent. At 37°C, T. marneffei grows as a uninucleate yeast that divides by fission. The yeast cells are the pathogenic form of this fungus. The protocols described here explain how to grow T. marneffei in the two vegetative growth forms in vitro, grow yeast cells inside mammalian macrophages, produce conidial stocks, and store strains both short and long term. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of the vegetative hyphal form on solid medium Alternate Protocol 1: Growth of the vegetative hyphal form in liquid suspension Basic Protocol 2: Growth of the vegetative yeast form on solid medium Alternate Protocol 2: Growth of the vegetative yeast form in liquid suspension Basic Protocol 3: Growth for production of dormant conidia Support Protocol: Preparation of Miracloth filter tubes Basic Protocol 4: Growth of Talaromyces marneffei in mammalian macrophages Basic Protocol 5: Storage of Talaromyces marneffei strains Alternate Protocol 3: Lyophilization of Talaromyces marneffei strains.

Kesesuaian Pemeriksaan jamur antara pewarnaan periodic acid schiff(PAS) dan KOH pada flour albus ibu hamil di RSUD dr Soetomo Surabaya

Background: Candidiasis vaginalis is a form of vaginal infection by Candida spp, estimated to occur in women of childbearing age in 70-75% of the cases. Clinical history, symptoms and laboratory examinations are very important to diagnose candidiasis vaginalis. Vaginal discharge examination can be done by direct or wet, and culture examination. Microscopic examination can be used to prove the existence of yeast form of Candida. This method is simple, can be applied in clinical microbiology laboratory with limited resources and is considered effective because it is cheap and results are obtained in a short time compared to culture. Detection of fungi by Periodic Acid Schiff (PAS) staining is visually better than KOH. The aim of this study is to analyze the concordance between Periodic Acid Schiff (PAS) staining and KOH method for fungal detection in flour albus from pregnant women.
 Method: This research is a descriptive observational study to figure out the suitability of Periodic Acid Schiff (PAS) staining and KOH method for fungal detection in flour albus from pregnant women with cross sectional approach. Samples were collected in the form of 30 vaginal discharge specimens taken from pregnant women and sent to the Clinical Microbiology Laboratory Dr. Soetomo Surabaya. The study was conducted in July - August 2019.
 Result: There was a significant difference in fungal detection between Periodic Acid Schiff (PAS) staining (56.7%, 17 samples) and 20% KOH method (6 samples) out of 30 samples (p=0.017). The concordance between Periodic Acid Schiff (PAS) staining and KOH method for fungal detection in flour albus from pregnant women is low (Kappa=0.321).
 Conclusion: Periodic Acid Schiff (PAS) staining detects more fungi when compared to KOH, and therefore is considered as a better method.
 Keywords: Flour albus, pregnancy, Periodic Acid Schiff (PAS), KOH.

Details of the Conformational Cycle of Hsp90 Probed using Optical Tweezers

Heat shock protein 90 (Hsp90) is an ATP-driven molecular chaperone which is essential in eukaryotes. It functions as a part of a number of complexes with many different co-chaperone proteins, which regulate its conformational cycle [1]. Using a custom-built single molecule optical trapping experimental setup, we have previously elucidated the mechanism by which this large protein folds [2], characterised the role of its flexible charged linker region [3], performed a detailed comparison of Hsp90 orthologs [4] and elucidated details of its dimerization dynamics [5]. Hsp90 is known to exist in two forms in yeast – the constitutively expressed Hsc82 and the form that is upregulated under cellular stress conditions, Hsp82 [6]. We recently performed a detailed comparison of these two isoforms, using a broad range of biophysical techniques to compare their function, structural properties and interactomes. Despite a 97% sequence similarity, we find significant differences in their stabilities, folding and conformational regulation [7]. We are currently working towards developing a complete quantitative description of the conformational cycle of Hsp82, as driven by co-chaperone proteins, nucleotides and clients using single molecule optical tweezers combined with standard biochemical techniques. [1] F. H. Schopf, M. M. Biebl & J. Buchner, Nat. Rev. Mol. Cell Biol. 18, 345 – 360, (2017). [2] M. Jahn, et al. Proc. Nat. Acad. Sci. 113, 1232–1237, (2016). [3] M. Jahn, et al. Proc. Nat. Acad. Sci. 111, 17881–17886, (2014). [4] M. Jahn and K. Tych, et al. Structure 26, 96 – 105, (2018). [5] K. Tych et al. J. Phys. Chem. B 122, 11373-11380 (2018). [6] K.A. Borkovic et al. Mol. Cell. Biol. 9, 3919 – 3930, (1989) [7] H. Girstmair and F. Tippel, et al. Nature Comms. 10, 1 – 15 (2019).

Blastomycosis

Blastomycosis is a serious fungal disease of humans and other mammals caused by environmentally acquired infection with geographically restricted, thermally dimorphic fungi belonging to the genus Blastomyces. The genetic and geographic diversity of these pathogens is greater than previously appreciated. In addition to Blastomyces dermatitidis and the cryptic species Blastomyces gilchristii, which cause blastomycosis in mid-western and various eastern areas of North America, atypical blastomycosis is occasionally caused by Blastomyces helicus in western parts of North America and Blastomyces percursus in Africa. Blastomycosis is acquired by inhalation of the conidia that are produced in the mold phase; in the lungs, temperature-dependent transformation occurs to the yeast phase. In this form, the organism is phagocytized by macrophages and can spread hematogenously to various organs causing disseminated infection. Pulmonary disease is most common and varies from mild, self-limited infection to severe, potentially fatal adult respiratory distress syndrome. Disseminated infection is manifested primarily by skin lesions, but many other organs can be involved. Diagnosis is established by growth of the organism in culture; however, a tentative diagnosis can be made quickly by histopathological identification of the classic yeast form in tissues or by finding Blastomyces antigen in urine or serum. Blastomycosis is treated initially with amphotericin B when the disease is severe, involves the central nervous system, or the host is immunosuppressed. Itraconazole is recommended for primary therapy in mild-to-moderate infection and for step-down therapy after initial amphotericin B treatment. Voriconazole and posaconazole can be used for patients in whom itraconazole is not tolerated.

Transcriptional control of hyphal morphogenesis in Candida albicans.

ABSTRACT Candida albicans is a multimorphic commensal organism and opportunistic fungal pathogen in humans. A morphological switch between unicellular budding yeast and multicellular filamentous hyphal growth forms plays a vital role in the virulence of C. albicans, and this transition is regulated in response to a range of environmental cues that are encountered in distinct host niches. Many unique transcription factors contribute to the transcriptional regulatory network that integrates these distinct environmental cues and determines which phenotypic state will be expressed. These hyphal morphogenesis regulators have been extensively investigated, and represent an increasingly important focus of study, due to their central role in controlling a key C. albicans virulence attribute. This review provides a succinct summary of the transcriptional regulatory factors and environmental signals that control hyphal morphogenesis in C. albicans.

The Effect of Oral Probiotic Streptococcus Salivarius K12 on Candida Albicans Biofilm Formation

Introduction: Oral cancer is the sixth most common cancer worldwide with Candida albicans infection being one of the aetiological factors for the disease. Meanwhile, Streptococcus salivarius K12 is an oral probiotic that is beneficial to the oral cavity. The objective of the present study is to determine the effect of S. salivarius K12 on C. albicans biofilm-forming ability with the hypothesis that S. salivarius K12 inhibits biofilm formation of C. albicans Materials and method: To assess the effect of S. salivarius K12 on C. albicans biofilm formation, S. salivarius K12, lab strain C. albicans MYA-4901 and clinical isolates from oral cancer, ALC2 and ALC3 were grown in both nutrient broth (NB) and RPMI. In a mono-species biofilm, 105 of C. albicans cells and 106 of S. salivarius K12 cells were grown separately in a 96-well plate. In contrast, both microorganisms were combined for polymicrobial biofilms with similar cell numbers as in mono-species. The biofilms were incubated for 72 hours at 37°C and the media were replenished every 24 hours. Finally, the crystal violet assay was conducted, and the optical density was measured at OD620nm. Results: Polymicrobial biofilms of C. albicans (MYA-4901 and ALC3) with S. salivarius K12 when grown in NB, exhibited a decrease by 64.5 ± 25.8% and 83.7 ± 5.4%, respectively when compared to the expected biofilms which were predominated by yeast form. Furthermore, polymicrobial biofilms of C. albicans (ALC2 and ALC3) with S. salivarius K12 showed a decrease by 62.5 ± 25.6% and 55.9 ± 17.1 %, respectively when compared to the expected biofilms when grown in RPMI that were predominated by hyphal form. Conclusion: S. salivarius K12 inhibited polymicrobial biofilms formation of C. albicans yeast and hyphal forms, thus supported the hypothesis that S. salivarius K12 inhibits biofilm formation of C. albicans.

Linking Sfl1 Regulation of Hyphal Development to Stress Response Kinases in Candida albicans.

Candida albicans is an important human pathogen responsible for causing both superficial and systemic infections. Its ability to switch from the yeast form to the hyphal growth form is required for its pathogenicity. Acidic pH inhibits hyphal initiation, but the nature of the mechanism for this inhibition is not completely clear. We show that acidic pH represses hyphal initiation independently of the temperature- and farnesol-mediated Nrg1 downregulation. Using a collection of transcription factor deletion mutants, we observed that the sfl1 mutant induced hyphae in acidic pH but not in farnesol at 37°C. Furthermore, transcription of hyphal regulators BRG1 and UME6 was not induced in wild-type (WT) cells but was induced in the sfl1 mutant during hyphal induction in acidic pH. Using the same screening conditions with the collection of kinase mutants, we found that deletions of the core stress response mitogen-activated protein (MAP) kinase HOG1 and its kinase PBS2, the cell wall stress MAP kinase MKC1, and the calcium/calmodulin-dependent kinase CMK1 allowed hyphal initiation in acidic pH. Furthermore, Hog1 phosphorylation induced by high osmotic stress also retarded hyphal initiation, and the effect was abolished in the sfl1 and three kinase mutants but was enhanced in the phosphatase mutant ptp2 ptp3 We also found functional associations among Cmk1, Hog1, and Sfl1 for cation stress. Our study results suggest that robust hyphal initiation requires downregulation of both Nrg1 and Sfl1 transcriptional repressors as well as timely BRG1 expression. Acidic pH and cationic stress retard hyphal initiation via the stress-responsive kinases and Sfl1.IMPORTANCECandida albicans is a commensal as well as a pathogen of humans. C. albicans is able to mount a cellular response to a diverse range of external stimuli in the host and switch reversibly between the yeast and hyphal growth forms. Hyphal development is a key virulence determinant. Here, we studied how C. albicans senses different environmental signals to control its growth forms. Our study results suggest that robust hyphal development requires downregulation of two transcriptional repressors, Nrg1 and Sfl1. Acidic pH or cationic stress inhibits hyphal formation via stress-responsive kinases and Sfl1.

Sanguinarine Inhibits Mono- and Dual-Species Biofilm Formation by Candida albicans and Staphylococcus aureus and Induces Mature Hypha Transition of C. albicans.

Previous studies have reported that sanguinarine possesses inhibitory activities against several microorganisms, but its effects on mono- and dual-species biofilms of C. albicans and S. aureus have not been fully elucidated. In this study, we aimed to evaluate the efficacy of sanguinarine for mono- and dual-species biofilms and explore its ability to induce the hypha-to-yeast transition of C. albicans. The results showed that the minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC90) of sanguinarine against C. albicans and S. aureus mono-species biofilms was 4, and 2 μg/mL, respectively, while the MIC and MBIC90 of sanguinarine against dual-species biofilms was 8, and 4 μg/mL, respectively. The decrease in the levels of matrix component and tolerance to antibiotics of sanguinarine-treated mono- and dual-species biofilms was revealed by confocal laser scanning microscopy combined with fluorescent dyes, and the gatifloxacin diffusion assay, respectively. Meanwhile, sanguinarine at 128 and 256 μg/mL could efficiently eradicate the preformed 24-h biofilms by mono- and dual-species, respectively. Moreover, sanguinarine at 8 μg/mL could result in the transition of C. albicans from the mature hypha form to the unicellular yeast form. Hence, this study provides useful information for the development of new agents to combat mono- and dual-species biofilm-associated infections, caused by C. albicans and S. aureus.

Candida albicans Ubiquitin and Heat Shock Factor-Type Transcriptional Factors Are Involved in 2-Dodecenoic Acid-Mediated Inhibition of Hyphal Growth.

Cis-2-dodecenoic acid (i.e., Burkholderia cenocepacia Diffusible Signal Factor, BDSF), a signaling molecule produced by Burkholderia cenocepacia but not by Candida albicans, can prevent Candida albicans hyphal formation. The mechanism by which BDSF controls the morphological switch of C. albicans is still unknown. To address this issue, we used the cDNA microarray method to investigate the differential expression of genes in C. albicans in the presence and absence of BDSF. The microarray result indicated that 305 genes were significantly different in the expression level. This included the downregulation of 75 genes and the upregulation of 230 genes. Based on the microarray data, a mutant library was screened to search for genes, once mutated, conferred insensitivity to BDSF. The results showed that the repressors (Ubi4 and Sfl1 proteins) and the activator (Sfl2 protein) of filamentous growth are involved in the BDSF regulation of hyphal morphogenesis. Ubi4, an ubiquitin polypeptide that participates in ubiquitin-mediated protein turnover, is the protein required for the degradation of Sfl2. Sfl1 and Sfl2 proteins antagonistically control C. albicans morphogenesis. In the hyphal induction condition, the amount of Ubi4 and Sfl1 protein increased rapidly with the exogenous addition of BDSF. As a result, the protein level of the activator of filamentous growth, Sfl2, decreased correspondingly, thereby facilitating the C. albicans cells to remain in the yeast form.

Effects of farnesol and lyticase on the formation of Candida albicans biofilm.

Background and Aim:Candida albicans is a dimorphic fungus that has both yeast and filamentous forms. It is part of the normal flora in the oral and genital areas of mammals. One factor for the pathogenicity of C. albicans is its ability to switch from yeast to hyphae. The hyphal form adheres and penetrates tissues more readily than the yeast form and produces biofilms that are associated with chronic infection. Biofilms are protective niches that enable microorganisms to be more resistant to antibiotic treatment, thus allowing for persistent infection. The first stage in the transition from yeast to hyphae involves the formation of a germ tube, and this transition is triggered by interactions with host cells. Germ tube formation is dependent on serum, pH, temperature, and quorum-sensing molecules (QSMs). Farnesol, which is a QSM in C. albicans, can prevent yeast to hyphae conversion and inhibits the growth of fungal biofilm. Lyticase is a synergistic enzyme complex that catalyzes yeast cell lysis by b-1,3-glucanase and is a highly specific alkaline protease that produces protoplasts or spheroplasts. This study investigated the effect of farnesol and lyticase on the formation of C. albicans biofilms.Materials and Methods:C. albicans ATCC 2091 was cultivated on liquid and solid Sabouraud media. The presence of C. albicans was confirmed using HiCrome Candida Agar chromogenic medium. Enzyme activities were assayed using a HiCandida Identification Kit. The morphology and densitometry parameters of C. albicans biofilms were considered in the presence of farnesol (Sigma-Aldrich, Germany), lyticase (from Arthrobacter luteus; Sigma-Aldrich, Germany), and farnesol–lyticase.Results:This study shows that both farnesol and lyticase possess antifungal activity against C. albicans biofilms. A significant difference among treatment groups (p<0.05) was observed from strong biofilm production to medium and weak.Conclusion:Many studies have been devoted to the antimicrobial action of farnesol. Bacterial enzyme lyticase is also used to degrade fungal cell walls. Both molecules show substantial antifungal properties that are similar to the properties of modern antimycotics. The current study demonstrates that farnesol and lyticase can disrupt biofilm formation in C. albicans ATCC 2091, which is an effective biofilm producer.

Genetic regulation of the development of mating projections in Candida albicans

ABSTRACT Candida albicans is a major human fungal pathogen, capable of switching among a range of morphological types, such as the yeast form, including white and opaque cell types and the GUT (gastrointestinally induced transition) cell type, the filamentous form, including hyphal and pseudohyphal cell types, and chlamydospores. This ability is associated with its commensal and pathogenic life styles. In response to pheromone, C. albicans cells are able to form long mating projections resembling filaments. This filamentous morphology is required for efficient sexual mating. In the current study, we report the genetic regulatory mechanisms controlling the development of mating projections in C. albicans. Ectopic expression of MTLα1 in “a” cells induces the secretion of α-pheromone and promotes the development of mating projections. Using this inducible system, we reveal that members of the pheromone-sensing pathway (including the pheromone receptor), the Ste11-Hst7-Cek1/2 mediated MAPK signalling cascade, and the RAM pathway are essential for the development of mating projections. However, the cAMP/PKA signalling pathway and a number of key regulators of filamentous growth such as Hgc1, Efg1, Flo8, Tec1, Ume6, and Rfg1 are not required for mating projection formation. Therefore, despite the phenotypic similarities between filaments and mating projections in C. albicans, distinct mechanisms are involved in the regulation of these two morphologies.

The signaling mechanisms involved in the dimorphic phenomenon of the Basidiomycota fungus Ustilago maydis.

In the present manuscript, we describe the mechanisms involved in the yeast-to-hypha dimorphic transition of the plant pathogenic Basidiomycota fungus Ustilago maydis. During its life cycle, U. maydis presents two stages: one in the form of haploid saprophytic yeasts that divide by budding and the other that is the product of the mating of sexually compatible yeast cells (sporidia), in the form of mycelial dikaryons that invade the plant host. The occurrence of the involved dimorphic transition is controlled by the two mating loci a and b. In addition, the dimorphic event can be obtained in vitro by different stimuli: change in the pH of the growth medium, use of different carbon sources, and by nitrogen depletion. The presence of other factors and mechanisms may affect this phenomenon; among these, we may cite the PKA and MAPK signal transduction pathways, polyamines, and factors that affect the structure of the nucleosomes. Some of these factors and conditions may affect all these dimorphic events, or they may be specific for only one or more but not all the processes involved. The conclusion reached by these experiments is that U. maydis has constituted a useful model for the analysis of the mechanisms involved in cell differentiation of fungi in general.

Antifungal activity of silver salts of Keggin-type heteropolyacids against Sporothrix spp.

Sporotrichosis is a chronic and subacute mycosis causing epidemiological outbreaks involving sick cats and humans in southeastern Brazil. The systemic disease prevails in cats, and in humans, the symptoms are restricted to skin in immunocompetent individuals. Under these conditions, the prolonged treatment of animals and cases of recurrence justify the discovery of new treatments for sporotrichosis. This work addresses the antifungal activity of silver salts of Keggin-type heteropolyacid salts (Ag-HPA salts) such as Ag3[PW12O40], Ag6[SiW10V2O40], Ag4[SiW12O40] and Ag3[PMo12O40] and interactions with the antifungal drugs itraconazole (ITC), terbinafine (TBF) and amphotericin B (AMB) on the yeast and mycelia forms of Sporothrix spp. Sporothrix spp. yeast cells were susceptible to Ag-HPA salts at minimum inhibitory concentration (MIC) values ranging from 8 to 128 μg/mL. Interactions between Ag3[PW12O40] and Ag3[PMo12O40] with itraconazole and amphotericin B resulted in higher antifungal activity with a reduction in growth and melanization. Treated cells showed changes in cell membrane integrity, vacuolization, cytoplasm disorder, and membrane detachment. Promising antifungal activity for treating sporotrichosis was observed for the Ag-HPA salts Ag3[PMo12O40] and Ag3[PW12O40], which have a low cost, high yield and activity at low concentrations. However, further evaluation of in vivo tests is still required.

Spruce sugars and poultry hydrolysate as growth medium in repeated fed-batch fermentation processes for production of yeast biomass

The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for reducing dependency on animal and plant protein. Growth media composed of enzymatically saccharified sulfite-pulped spruce wood, enzymatic hydrolysates of poultry by-products and urea were used for the production of single-cell protein. Strains of three different yeast species, Cyberlindnera jadinii, Wickerhamomyces anomalus and Blastobotrys adeninivorans, were cultivated aerobically using repeated fed-batch fermentation up to 25 L scale. Wickerhamomyces anomalus was the most efficient yeast with yields of 0.6 g of cell dry weight and 0.3 g of protein per gram of glucose, with cell and protein productivities of 3.92 g/L/h and 1.87 g/L/h, respectively. Using the conditions developed here for producing W. anomalus, it would take 25 industrial (200 m3) continuously operated fermenters to replace 10% of the fish feed protein used in Norway.

Guinea pig seborrheic dermatitis model of Malassezia restricta and the utility of luliconazole.

Seborrheic dermatitis (SD) is a multifactorial disease in which Malassezia restricta has been proposed as the predominant pathogenic factor. However, experimental evidence supporting this hypothesis is limited. A guinea pig SD model using a clinical isolate of M. restricta was used to elucidate the pathogenicity of M. restricta. Also, the efficacy of 1% luliconazole (LLCZ) cream, a topical imidazole derivative, against M. restricta was compared with that of a 2% ketoconazole (KCZ) cream in the same guinea pig model. Dorsal skin hairs of guinea pig were clipped and treated with M. restricta by single or repeated inoculations without occlusion. Skin manifestations were examined macroscopically and histologically. A quantitative polymerase chain reaction (PCR) assay was also performed for mycological evaluation. An inflammatory response mimicking SD occurred after repeated as well as single inoculation but not in abraded skin. The inflammation score attained its maximum on day 11 and persisted until day 52. The yeast form of the fungal elements was distributed on the surface of stratum corneum and around the follicular orifices, and an epidermal and dermal histological reaction was observed. Application of 1% LLCZ or 2% KCZ cream significantly improved the skin manifestations and decreased the quantity of M. restricta rDNA in the skin lesions. The efficacy of topical antifungal drugs suggested that M. restricta is a pathogenic factor contributing to SD.

Biological and genomic analyses of a clinical isolate of Yarrowia galli from China.

Infections caused by emerging fungal pathogens represent a new threat to human health. The yeast Yarrowia (Candida) galli was first described from chicken breast and liver in 2004 and has occasionally been isolated in clinical settings. In this study, we present the first report of a Y. galli isolate from a face granuloma of a woman. Y. galli is unable to grow at human physiological temperature (37°C). Phenotypic analysis demonstrates that Y. galli can exist as several morphological types, namely fluffy, sticky, tight, and yeast forms, based on their cellular and colony appearances. Interestingly, Y. galli is able to undergo switching among different morphologies. These morphological changes are similar to the switching systems in pathogenic Candida species such as Candida albicans and Candida tropicalis. We further sequenced the genome of the Y. galli isolate. A comparative analysis with pathogenic yeast species indicated that a set of lipid metabolism genes were enriched in Y. galli. Domain enrichment analysis demonstrated that, similar to Candida clade species, the genome of Y. galli maintained several gene families required for virulence. Our biological and genomic analyses provide new insights into the understanding of the biology of Y. galli as either an environmental isolate or a potential human pathogen.