Pathogenic Yeast Research Articles

Improving homology-directed repair by small molecule agents for genetic engineering in unconventional yeast?-Learning from the engineering of mammalian systems.

The ability to precisely edit genomes by deleting or adding genetic information enables the study of biological functions and the building of efficient cell factories. In many unconventional yeasts, such as those promising new hosts for cell factory design but also human pathogenic yeasts and food spoilers, this progress has been limited by the fact that most yeasts favour non-homologous end joining (NHEJ) over homologous recombination (HR) as a DNA repair mechanism, impairing genetic access to these hosts. In mammalian cells, small molecules that either inhibit proteins involved in NHEJ, enhance protein function in HR, or arrest the cell cycle in HR-dominant phases are regarded as promising agents for the simple and transient increase of HR-mediated genome editing without the need for a priori host engineering. Only a few of these chemicals have been applied to the engineering of yeast, although the targeted proteins are mostly conserved, making chemical agents a yet-underexplored area for enhancing yeast engineering. Here, we consolidate knowledge of the available small molecules that have been used to improve HR efficiency in mammalian cells and the few ones that have been used in yeast. We include available high-throughput-compatible NHEJ/HR quantification assays that could be used to screen for and isolate yeast-specific inhibitors.

Identification of heparin-binding proteins expressed on Trichosporon asahii cell surface.

Trichosporon asahii is a pathogenic yeast that cause trichosporonosis. T. asahii exhibits several colony morphologies, such as white (W)- or off-white (O)-type, which may affect virulence. In this study, we compared the expression pattern of heparin-binding proteins in various colony morphologies and identified heparin-binding protein in T. asahii. Surface plasmon resonance analysis revealed that cell surface molecules attached more strongly to heparin in W- than O-type cells. We purified and identified a heparin-binding protein strongly expressed in W-type cells using heparin-Sepharose beads, named it heparin-binding protein 1 (HepBP1), and expressed Flag-tagged HepBP1 in mammalian cells. The heparin-binding ability of Flag-tagged HepBP1 was confirmed by pulldown assay using heparin-Sepharose beads. Thus, HepBP1 is a heparin-binding protein on T. asahii cell surface. These results suggest that several T. asahii cell surface proteins interact with glycosaminoglycans; therefore, they could contribute to infection.

A Quick reCAP: Discovering Cryptococcus neoformans Capsule Mutants.

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause severe meningoencephalitis in immunocompromised hosts and is a leading cause of death in HIV/AIDS patients. This pathogenic yeast is surrounded by a polysaccharide capsule that is critical for virulence and plays important roles in host-pathogen interactions. Understanding capsule biosynthesis is therefore key to defining the biology of C. neoformans and potentially discovering novel therapeutic targets. By exploiting methods to identify mutants deficient in capsule, June Kwon-Chung and other investigators have discovered numerous genes involved in capsule biosynthesis and regulation. Successful approaches have incorporated combinations of techniques including mutagenesis and systematic gene deletion; complementation and genetic screens; morphological examination, physical separation, and antibody binding; and computational modeling based on gene expression analysis. In this review, we discuss these methods and how they have been used to identify capsule mutants.

Chelation of Mitochondrial Iron as an Antiparasitic Strategy.

Iron, as an essential micronutrient, plays a crucial role in host-pathogen interactions. In order to limit the growth of the pathogen, a common strategy of innate immunity includes withdrawing available iron to interfere with the cellular processes of the microorganism. Against that, unicellular parasites have developed powerful strategies to scavenge iron, despite the effort of the host. Iron-sequestering compounds, such as the approved and potent chelator deferoxamine (DFO), are considered a viable option for therapeutic intervention. Since iron is heavily utilized in the mitochondrion, targeting iron chelators in this organelle could constitute an effective therapeutic strategy. This work presents mitochondrially targeted DFO, mitoDFO, as a candidate against a range of unicellular parasites with promising in vitro efficiency. Intracellular Leishmania infection can be cleared by this compound, and experimentation with Trypanosoma brucei 427 elucidates its possible mode of action. The compound not only affects iron homeostasis but also alters the physiochemical properties of the inner mitochondrial membrane, resulting in a loss of function. Furthermore, investigating the virulence factors of pathogenic yeasts confirms that mitoDFO is a viable candidate for therapeutic intervention against a wide spectrum of microbe-associated diseases.

Expansion of the antifungal activities through in silico docking study of compounds from Albizia lebbeck fruits

The development of new computational and experimental techniques that are efficient, fast, and accurate has become a priority for researchers in all domains around the world. The present manuscript deals with the computational investigation of quercitrin (1), lebbeckisoetin A (2), quercetin-3-O-_β_-D-glucopyranoside (3), (_E_)-_p_-coumaric acid (4), eugenol (5), eugenol, 1-acetate (6), chiakine (7), hexancosan-1',26'-dioate of bis[(2_S_), 2,3-dihydroxypropyl] (8), oleanolic acid (9), betulin (10), hopan-29-ol (11), hopan-30-ol (12), 22-hydroxyhopan-3-ol (13), and lupeol (14). These compounds were all isolated from the fruit of _Albizia lebbeck_. Only lebbeckisoetin A (2) and chiakine (7) were previously evaluated for their experimental antimicrobial activities, which both revealed potent antifungal activities. The virtual screening of the antifungal activity was performed on Maestro Schrӧdinger software using sterol 14-alpha demethylase (CYP51) from _Candida albicans_ in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1) and sterol 14-alpha demethylase (CYP51) from the pathogenic yeast _Candida albicans_ in complex with the antifungal drug posaconazole (PDB Id: 5FSA). This is the first docking study of natural compounds using these 5TZ1 and 5FSA as proteins. Compounds (1-10) reveal interesting binding strength with both 5TZ1 and 5FSA proteins, with a docking score ranging from -7.892 to -5.256, supporting the experimental results. In addition, compounds (1-9) were mostly active due to the formation of π-π interactions, H-bonds, and hydrophobic interactions, as well as π-cation and salt bridge interactions with both 5TZ1 and 5FSA.

Production and evaluation of novel functional cream cottage cheese fortified with bovine colostrum and probiotic bacteria

Nutrient-dense colostrum can be employed as a functional food ingredient. This work aimed to produce novel functional probiotic Cream cottage cheese (FPC) using probiotic (ABT) culture and bovine colostrum powder (BCP) at levels of 1, 2, and 3%. Physicochemical and functional properties (antioxidant activity, fatty acid profile, and antibacterial activity) were analyzed. The outcome revealed that hardness, cohesiveness, and gumminess were increased while springiness and chewiness were decreased for the treated cheeses. In FPC, medium-chain fatty acids were the predominant forms, followed by short- and long-chain fatty acids, polyunsaturated (PUFA), and small amounts of monounsaturated (MUFA). The antioxidant activity of all the cheese samples was significantly (P < 0.05) increased by increasing the quantity of colostrum powder and lengthening storage time. Color parameters were influenced by enrichment with BCP, whether in fresh or stored samples. With the addition of BCP, the growth of lactic acid bacteria and Bifidobacteria was enhanced, whereas that of pathogenic bacteria, mold and yeast, and coliform groups was inhibited. Cheeses fortified with 2% BCP had significantly higher score values than those in the other treatments. Therefore, it could be concluded that cottage cheese fortified with 2% BCP has high nutritional value and health benefits.

Expansion of the antifungal activities through in silico docking study of compounds from Albizia lebbeck fruits

The development of new computational and experimental techniques that are efficient, fast, and accurate has become a priority for researchers in all domains around the world. The present manuscript deals with the computational investigation of quercitrin (1), lebbeckisoetin A (2), quercetin-3-O-_β_-D-glucopyranoside (3), (_E_)-_p_-coumaric acid (4), eugenol (5), eugenol, 1-acetate (6), chiakine (7), hexancosan-1',26'-dioate of bis[(2_S_), 2,3-dihydroxypropyl] (8), oleanolic acid (9), betulin (10), hopan-29-ol (11), hopan-30-ol (12), 22-hydroxyhopan-3-ol (13), and lupeol (14). These compounds were all isolated from the fruit of _Albizia lebbeck_. Only lebbeckisoetin A (2) and chiakine (7) were previously evaluated for their experimental antimicrobial activities, which both revealed potent antifungal activities. This theoretical study was undertaken in the framework of better understanding the experimental results at the atomic level and to expand the antimicrobial assays of the other non-tested compounds. The virtual screening of the antimicrobial activity was performed on Maestro Schrӧdinger 4.2.1 software using the crystal structures of sterol 14-alpha demethylase (CYP51) from _Candida albicans_ in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1) and the crystal structure of sterol 14-alpha demethylase (CYP51) from the pathogenic yeast _Candida albicans_ in complex with the antifungal drug posaconazole (PDB Id: 5FSA). This is the first docking study of natural compounds using these 5TZ1 and 5FSA as proteins. Compounds (1-10) reveal interesting binding strength with both 5TZ1 and 5FSA proteins, with a docking score ranging from -7.892 to -5.256, supporting the experimental results. In addition, compounds (1-9) were mostly active due to the formation of π-π interactions, H-bonds, and hydrophobic interactions, as well as π-cation and salt bridge interactions, with both 5TZ1 and 5FSA.

Modulators of Candida albicans Membrane Drug Transporters: A LucrativePortfolio for the Development of Effective Antifungals.

The escalating prevalence of membrane drug transporters and drug efflux pumps in pathogenic yeast like Candida albicans necessitates a comprehensive understanding of their roles in MDR. The overexpression of drug transporter families, ABC and MFS, implicated in MDR through drug efflux and poses a significant challenge in the diagnosis and treatment of fungal infection. Various mechanisms have been proposed for MDR; however, the upregulation of ABC and MFS superfamily transporters is most noticeable in MDR. The direct inhibition of these transporters seems an efficient strategy to overcome this problem. The goal of the article is to present an overview of the prospect of utilizing these modulators of C. albicans drug transports as effective antifungal molecules against MDR addressing a critical gap in the field. The review tries to address to prevent drug extrusion by modulating the expression of drug transporters of C. albicans. The review discussed the progress in identifying potent, selective, and non-toxic modulators of these transporters to develop some effective antifungals and overcome MDR. We reviewed major studies in this area and found that recent work has shifted toward the exploration of natural compounds as potential modulators to restore drug sensitivity in MDR fungal cells. The focus of this review is to survey and interpret current research information on modulators of C. albicans drug transporters from natural sources emphasizing those compounds that are potent antifungal agents.

Antifungal susceptibility profile and local epidemiological cut-off values of Yarrowia (Candida) lipolytica: an emergent and rare opportunistic yeast.

Yarrowia lipolytica, also known as Candida lipolytica, is an emerging opportunistic "rare pathogenic yeast". Due to the limited data on its antifungal susceptibility, the clinical treatments become challenging. Based on the China Hospital Invasive Fungal Surveillance Network (2009-2022), we conducted a comprehensive multi-method study on clinical isolates from various central hospitals. This study is currently the largest study carried out to assess the antifungal susceptibility of Y. lipolytica. It is also the first to establish local epidemiological cut-off values (L-ECOFFs), identify its ERG11 mutations, and assess the consistency between the three prevalent commercial antifungal susceptibility testing methods and the broth microdilution method. We recommend the Sensititre YeastOne as the best option for antifungal susceptibility testing for Y. lipolytica, followed by the ATB FUNGUS 3. Nevertheless, practitioners should use the MIC test strip with discretion.

Analysis of the culturable gut yeast microbiota of dogs with digestive disorders

Despite the increasing interest in studying the gut mycobiota of dogs, the association between fungal colonization and the development of digestive disorders in this species remains largely understudied. On the other hand, the high prevalence of antifungal-resistant yeasts detected in previous studies in samples from animals represents a major threat to public health. We analyzed the presence of culturable yeasts in 112 rectal swab samples obtained from dogs with digestive disorders attended in a veterinary teaching hospital. Our results revealed that Malassezia pachydermatis was frequently isolated from the studied dog population (33.9% of samples), and that the isolation of this yeast was significantly associated to the age of animals, but not to their sex, disease group, or the presence of vomits and/or diarrhea. In contrast, other yeast species were less prevalent (17.9% of samples in total), and their isolation was not significantly associated to any variable included in the analysis. Additionally, we observed that 97.5% of the studied M. pachydermatis isolates (n = 158, 1–6 per positive episode) displayed a minimum inhibitory concentration (MIC) value >4 μg/ml to nystatin, 31.6% had a MIC ≥32 μg/ml to fluconazole, and 27.2% had a MIC >4 μg/ml to amphotericin B. The antifungal susceptibility profiles of non-Malassezia (n = 43, 1–7 per episode) were more variable and included elevated MIC values for some antifungal-species combinations. These results confirm that the intestine of dogs is a reservoir of opportunistic pathogenic yeasts and suggest that the prevalence of M. pachydermatis colonization depends more on the age of animals than on any specific digestive disorder.

Heat Shock Protein SSA1 Enriched in Hypoxic Secretome of Candida albicans Exerts an Immunomodulatory Effect via Regulating Macrophage Function.

Candida albicans is an opportunistic pathogenic yeast that can survive in both normoxic and hypoxic environments. The involvement of C. albicans secretome on host biological processes has been demonstrated. However, the immunoregulatory function of C. albicans secretome released under hypoxic condition remains unclear. This study demonstrated the differences in cytokine responses and protein profiles between secretomes prepared under normoxic and hypoxic conditions. Furthermore, the immunoregulatory effects of heat shock protein SSA1(Ssa1), a protein candidate enriched in the hypoxic secretome, were investigated. Stimulation of mouse bone marrow-derived macrophages (BMMs) with Ssa1 resulted in the significant production of interleukin (IL)-10, IL-6, and tumor necrosis factor (TNF)-α as well as the significant expression of M2b macrophage markers (CD86, CD274 and tumor necrosis factor superfamily member 14), suggesting that C. albicans Ssa1 may promote macrophage polarization towards an M2b-like phenotype. Proteomic analysis of Ssa1-treated BMMs also revealed that Ssa1 reduced inflammation-related factors (IL-18-binding protein, IL-1 receptor antagonist protein, OX-2 membrane glycoprotein and cis-aconitate decarboxylase) and enhanced the proteins involved in anti-inflammatory response (CMRF35-like molecule 3 and macrophage colony-stimulating factor 1 receptor). Based on these results, we investigated the effect of Ssa1 on C. albicans infection and showed that Ssa1 inhibited the uptake of C. albicans by BMMs. Taken together, our results suggest that C. albicans alters its secretome, particularly by promoting the release of Ssa1, to modulate host immune response and survive under hypoxic conditions.

Digestive processes and microbiocenosis of intestinal contents in sheep when using a «protected» form of betaine in nutrition

In the process of maintaining the metabolic processes of the gastrointestinal tract of ruminants, an important link is to ensure a complete feeding diet. The study of the use of betaine supplements in a form "protected" from the indirect effects of the microflora of the rumen showed positive dynamics in relation to the digestive processes, the microbial composition of the rumen and the intestines of sheep. In the conditions of the physiological yard of the Federal Science Center for Animal Husbandry named after Academy Member L.K. Ernst conducted a physiological experiment on ramshanks of crossbreeds of the Romanov breed and katadins, aged 8-9 months, with chronic scar fistulas according to Basov (n=3). In the control period, the animals received the main diet, in the experimental 1 – the main diet + unprotected betaine at a dosage of 0.05 g / kg of live weight, in the 2nd experimental period a "protected" betaine supplement at a dosage of 0.05 g / kg of live weight. The duration of each period was 30 days. During the accounting period, average daily stool samples were taken according to the generally accepted method. To study cicatricial digestion, at the end of each period, samples of the contents were taken from animals through the fistula of the scar by Basov. As a result of the use of both the "protected" and native forms of betaine, the mass of microorganisms of the scar content became higher than in the control period. When the "protected" form of betaine was added to the diet of animals, the overall digestibility of feed nutrients tended to improve. In the 2nd experimental period, compared with the control, the level of bifidobacteria in the large intestine of sheep increased by 1.63 CFU/g, the level of pathogenic yeast fungi decreased several times.

Synthesis and characterization of silver nanoparticles loaded with carboplatin as a potential antimicrobial and cancer therapy

In recent studies with silver nanoparticles, it has been reported that the use of nanoparticles in carrier drug systems increases tumor suppression and reduces drug-related side effects. At the same time, the combination of traditional medicine with nanotechnology provides the opportunity to develop new antimicrobial agents. The aim of this study was to determine the anticancer, antimicrobial activities and pro-apoptotic effects of silver nanoparticles (AgNPs), and carboplatin-loaded silver nanoparticles (AgNPs-Car). Characterization studies of the synthesized nanoparticles were carried out by DLS, EDX-STEM, and FTIR analysis. The antiproliferative and pro-apoptotic effects of these molecules were evaluated using XTT and Annexin V, respectively. MIC (Minimum Inhibitory Concentration) test was used to determine the antimicrobial activity. The anticancer activity of the AgNPs-Car was high in MCF-7 (human breast adenocarcinoma), A549 (human lung carcinoma), and C6 (brain glioma) cells. The cell group with the most effective selective cytotoxic activity was C6 cells. It was also shown that AgNPs-Car and AgNPs induced DNA fragmentation eventually increasing apoptosis of cells. The antimicrobial activity of AgNPs and AgNPs-Car was evaluated on Gram-positive and Gram-negative pathogenic microorganisms and yeast fungi. Among the nanomaterials that reached effective MIC values according to reference sources, AgNPs-Car achieved better results. As a result, AgNPs-Car was found to be very successful in targeting C6 glioma cells by facilitating cell entry of the drug. In addition, their anticancer activity on MCF-7 and A549 cells was high and their toxicity was low. Silver nanoparticles are preferred for creating a better drug carrier system because of their qualitative properties and effects. Therefore, it is an interesting field for research on targeting cancer cells and pathogenic microorganisms.

Chemical constituents and in vitro antimicrobial activity of rhizome essential oils of Zingiber densissimum S.Q.Tong & Y.M.Xia and Kaempferia laotica Gagnep. growing wild in Vietnam

Zingiber densissimum S.Q.Tong & Y.M.Xia and Kaempferia laotica Gagnep., belong to the Zingiberaceae family, which were found in Vietnam and Laos in recent years. To date, there has been very limited information on their phytochemical profiles and pharmacological activities. The present study was conducted to give insights into the chemical composition of the essential oils isolated from two plants and their antimicrobial activities. Gas Chromatographic–Mass Spectral (GC-MS) analysis was utilized to identify the chemical composition of the rhizome essential oils of Z. densissimum and K. laotica. The antimicrobial activities were evaluated on three Gram-positive bacterial strains, three Gram-negative bacterial strains, and a pathogenic yeast. The GC-MS analysis showed that β-pinene (38.36%), β-phellandrene (26.85%), and α-pinene (13.31%) were the most dominant components in Z. densissimum rhizome essential oil, while K. laotica rhizome essential oil was found to contain mainly camphene (23.44%), α-pinene (10.69%), 3-carene (8.60%) and β-pinene (6.85%). As for the antimicrobial activity test results, the rhizome essential oil of Z. densissimum displayed potent activities against Candida albicans (MIC value = 2 μg/mL), Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli (MIC values = 8 μg/ mL). Meanwhile, the rhizome essential oil of K. laotica showed weaker activities against all investigated microbial strains (MIC values ranging from 32 to 256 μg/mL). The study results indicated the chemical constituents of the essential oils prepared from these plants. The two essential oil samples were also shown to exhibit potential antimicrobial activities against several pathogenic bacterial and fungal strains.

LAMinar Flow: Sterol Transport in a Pathogenic Yeast.

Cryptococcus neoformans is an important fungal pathogen, responsible for over 140,000 deaths per year worldwide. Like other yeasts, C. neoformans relies on ergosterol as its major membrane sterol and carefully regulates its synthesis and distribution. Ergosterol is also targeted by two of the three compound classes currently used to treat cryptococcal infection. We recently reported the discovery and characterization in C. neoformans of a single retrograde ergosterol transporter of the LAM family, Ysp2. Here we review these findings and discuss directions for future research, including the connections between processes that are perturbed by the absence of Ysp2 (which also abrogates cryptococcal virulence) and possible roles for Ysp2 and other, as yet unknown, lipid transport proteins in this organism.

Synthesis, characterisation and antimicrobial activity of supramolecular cobalt-peptide conjugates.

Herein, we describe the synthesis and characterisation of four new supramolecular cobalt conjugates of antimicrobial peptides functionalised with terpyridine ligands (L). Peptides were chosen based on the well-established arginine-tryptophan (RW)3 motif, with terpyridine-derivatized lysine (Lys(tpy)) added to the sequence, or replacing tryptophan residues. Self-assembly of the antimicrobial peptides with Co(BF4)2·6H2O formed exclusively CoL2 dimers (for peptides with one tpy ligand each) and Co2L4 metallo-macrocycles (for peptides with two tpy ligands for each peptide), which could be 'locked' by oxidation of Co(+II) to Co(+III) with ammonium ceric nitrate. The Co-peptide complexes were characterised by mass spectrometry and in solution by NMR spectroscopy, including 2D diffusion ordered NMR spectroscopy (DOSY) which confirmed the proposed stoichiometries. The antimicrobial activity of the novel peptides and their metallo-supramolecular assemblies was investigated by determination of their minimal inhibitory concentration (MIC) against a panel of Gram-positive and Gram-negative bacteria. Complexation with cobalt increases the activity of the peptides in almost every case. Most of the new metal-peptide conjugates showed good activity against Gram-positive bacteria, including a multi-resistant S. aureus strain and the opportunistic pathogenic yeast C. albicans (down to 7 μmol l-1 for the most active Co2L4 derivate), a value that is increased five-fold compared to the lysine-derivatized peptide ligand alone. Interestingly, conjugates of the CoL2 type also showed decent activity against Gram-negative bacteria including the WHO-flagged problematic A. baumannii strain (down to 18 μmol l-1 for the most active derivative).

Two promising Bacillus-derived antifungal lipopeptide leads AF4 and AF5 and their combined effect with fluconazole on the in vitro Candida glabrata biofilms.

Introduction: Candida species are endowed with the ability to produce biofilms, which is one of the causes of pathogenicity, as biofilms protect yeasts from antifungal drugs. Candida glabrata (Nakaseomyces glabrata) is one of the most prevalent pathogenic yeasts in humans and a biofilm producer. Methods: The study was aimed at evaluating the combined effects of two highly promising antifungal biomolecules (AF4 and AF5) lipopeptide in nature, chromatographically purified to homogeneity from Bacillus subtilis (B. subtilis) and the standard antifungal fluconazole (at different concentrations) to demonstrate C. glabrata biofilm formation inhibition. Biofilm production and inhibition were evaluated by quantification of the biofilm biomass and metabolic activity using crystal violet (CV) staining and XTT reduction assays, respectively. Microscopic techniques such as confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM) were employed to visualize biofilm formation and inhibition. Results and Discussion: Compared to untreated and fluconazole-treated biofilms, an enhanced in vitro anti-biofilm effect of the antifungal lipopeptides AF4/AF5 alone and their combinations with fluconazole was established. The lipopeptides AF4/AF5 alone at 8 and 16μg/mL exhibited significant biomass and metabolic activity reductions. SEM and CSLM images provided evidence that the lipopeptide exposure results in architectural alterations and a significant reduction of C. glabrata biofilms, whereas (2', 7'-dichlorofluorescin diacetate (DCFDA) and propidium iodide (PI) analyses showed reactive oxygen species (ROS) generation along with membrane permeabilization. The estimation of exopolysaccharides (EPS) in AF4/AF5-treated biofilms indicated EPS reduction. The combinations of fluconazole (64/128μg/mL) and AF4/AF5 lipopeptide (16μg/mL) were found to significantly disrupt the mature (24h) biofilms as revealed by CSLM and SEM studies. The CSLM images of biofilms were validated using COMSTAT. The FTIR-analyses indicate the antibiofilm effects of both lipopeptides on 24h biofilms to support CSLM and SEM observations. The combinations of fluconazole (64/128μg/mL) and AF4/AF5 lipopeptide were found to disrupt the mature biofilms; the study also showed that the lipopeptides alone have the potentials to combat C. glabrata biofilms. Taken together, it may be suggested that these lipopeptide leads can be optimized to potentially apply on various surfaces to either reduce or nearly eradicate yeast biofilms.

Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerance.

Development of resistance and tolerance to antifungal drugs in Candida albicans can compromise treatment of infections caused by this pathogenic yeast species. The uniquely expanded C. albicans TLO gene family is comprised of 14 paralogous genes which encode Med2, a subunit of the multiprotein Mediator complex which is involved in the global control of transcription. This study investigates the acquisition of fluconazole tolerance in a mutant in which the entire TLO gene family has been deleted. This phenotype was reversed to varying degrees upon reintroduction of representative members of the alpha- and beta-TLO clades (i.e. TLO1 and TLO2), but not by TLO11, a gamma-clade representative. Comparative RNA sequencing analysis revealed changes in the expression of genes involved in a range of cellular functions, including ergosterol biosynthesis, mitochondrial function, and redox homeostasis. This was supported by the results of mass spectrometry analysis, which revealed alterations in sterol composition of the mutant cell membrane. Our data suggest that members of the C. albicans TLO gene family are involved in the control of ergosterol biosynthesis and mitochondrial function and may play a role in the responses of C. albicans to azole antifungal agents.

Use of the Fluorescent Dye Thioflavin T to Track Amyloid Structures in the Pathogenic Yeast Candida albicans.

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm. Key features • Study of amyloid properties of fungal proteins. • Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy. • Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation. • Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.

Measuring Replicative Lifespan in Cryptococcus neoformans.

Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.