Ncs2* mediates in vivo virulence of pathogenic yeast through sulphur modification of cytoplasmic transfer RNA.

Fungal pathogens threaten ecosystems and human health. Understanding the molecular basis of their virulence is key to develop new treatment strategies. Here, we characterize NCS2*, a point mutation identified in a clinical baker's yeast isolate. Ncs2 is essential for 2-thiolation of tRNA and the NCS2* mutation leads to increased thiolation at body temperature. NCS2* yeast exhibits enhanced fitness when grown at elevated temperatures or when exposed to oxidative stress, inhibition of nutrient signalling, and cell-wall stress. Importantly, Ncs2* alters the interaction and stability of the thiolase complex likely mediated by nucleotide binding. The absence of 2-thiolation abrogates the in vivo virulence of pathogenic baker's yeast in infected mice. Finally, hypomodification triggers changes in colony morphology and hyphae formation in the common commensal pathogen Candida albicans resulting in decreased virulence in a human cell culture model. These findings demonstrate that 2-thiolation of tRNA acts as a key mediator of fungal virulence and reveal new mechanistic insights into the function of the highly conserved tRNA-thiolase complex.

How fungal multidrug transporters mediate hyper resistance through DNA amplification and mutation.

A significant portion of clinically observed antifungal resistance is mediated by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transport pumps that reside in the plasma membrane. We review the mechanisms responsible for this phenomenon. Hyper resistance is often brought about by several kinds of DNA amplification or by gain-of-function mutations in a variety of transcription factors. Both of these result in overexpression of ABC and MFS transporters. Recently, however, several additional modes of resistance have been observed. These include mutations in non-conserved nucleotides leading to altered mRNA stability and a mutation in yeast transporter Pdr5, which improves cooperativity between drug-binding sites.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Incidence of Saccharomyces cerevisiae fungemia in hospitalised patients administered Saccharomyces boulardii probiotic.

BACKGROUND: Bloodstream infection is an untoward effect of probiotic administration described by case reports and a cited reason to avoid using in the critically ill. The incidence rate of bloodstream infection in a population administered probiotics remains unknown. METHODS: A retrospective observational analysis of incident Saccharomyces cerevisiae fungemia in a population of hospitalised patients administered Saccharomyces boulardii for primary prevention of hospital-onset Clostridioides difficile infection. Adult patients admitted to an inpatient medical unit for 48-h or more between January 1, 2016 and December 31, 2019 are included. Facility medication administration records and microbiology records were evaluated for S boulardii probiotic administration and incidence of S cerevisiae positive blood cultures. Microbiologic identification methods were unable to distinguish S cerevisiae from S boulardii. RESULTS: Administration of S boulardii probiotic occurred in 16,404 of 46,729 patients analysed. S cerevisiae fungemia was identified in 18 probiotic recipients (0.11%). The observed incidence of fungemia attributable to S boulardii administration is 1.70 cases per 10,000 patient-days. Central-line days numbered 52,949 yielding an incidence of 0.26 cases of S cerevisiae per 1,000 central-line days. Intensive care unit admission was significantly associated with an increase in the risk of S cerevisiae (OR 6.55, CI 2.28-18.87), incidence rate of 0.47 cases per 1,000 patient-days. CONCLUSION: The risk of bloodstream infection as a result of S boulardii probiotic use appears restricted to S boulardii recipients. The risk for probiotic-related bloodstream infection does not appear greater than the risk of any hospital-acquired bloodstream infection both inside and outside of the intensive care unit.

Saccharomyces cerevisiae fungemia: Risk factors, outcome and links with S. boulardii-containing probiotic administration.

Saccharomyces cerevisiae fungemia: risk factors, outcome and links with S. boulardii-containing probiotic administration. OBJECTIVE: The aim of our study was to review cases of S. cerevisiae fungemia along with the corresponding risk factors (including S. boulardii probiotic intake), treatment and outcomes. PATIENTS AND METHODS: Retrospective study (2005-2017) of S. cerevisiae fungemia. All the data were extracted from medical files. RESULTS: We identified 10 patients with S. cerevisiae fungemia. Mean age was 59.4 years (range 21-88). Four fifths (80%) were on total parenteral or enteral nutrition, 70% had a central venous line, and 30% were admitted in an Intensive Care Unit (ICU). S. boulardii-containing probiotic prescription was identified in 6 subjects. Three patients with no risk factors such as ICU or central venous catheter were 80 years old or more. Mortality rate was 50%. CONCLUSION: S. cerevisiae fungemia is a rare but life-threatening infection, associated with intake of probiotics containing S. boulardii. Besides classical risk factors, older age should be a contraindication for these probiotics.

Antifungal activity of Saccharomyces cerevisiae and assessment of ochratoxigenic load on currants by means of Real Time PCR.

Currants are prone to contamination by ochratoxin during cultivation, processing and storage conditions. Saccharomyces cerevisiae is considered to be among the main species of grape yeast flora able to control antagonistic fungi. In this study, the potential of S. cerevisiae Y33 was investigated to inhibit the growth of several fungal species indigenous to the microbiota of grapes. Moreover, the efficacy of this yeast species was investigated to inhibit OTA by toxin producing fungi both in vitro and in situ. For this purpose thirty-five different fungal species, belonging to the genera Aspergillus, Penicillium, Cladosporium, Fusarium and Alternaria interacted in vitro with S. cerevisiae on Malt Extract agar plates, stored at 25 °C for 14 days. Results showed that the highest OTA producer A. carbonarius F71 was inhibited more than 99% from day 7, in contrast to A. niger strains that presented enhanced OTA production at day 14 due to interaction with S. cerevisiae Y33. Additionally, the antifungal potential of the selected yeast was also studied in situ on currants subjected to different treatments and stored at 25 °C for 28 days. Microbiological analysis was undertaken for the enumeration of the bacterial and fungal flora, together with OTA determination at 7 and 21 days. To quantify A. carbonarius on all treated currant samples, molecular analysis with Real Time PCR was employed. A standard curve was prepared with A. carbonarius DNA. The efficiency of the curve was estimated to 10.416, the slope to -3.312 and the range of haploid genome that could be estimated was from 1.05 to 105∙105. The amount of A. carbonarius DNA in all treated currants samples, where the fungus was positively detected, ranged from as low as 0.08 to 562 ng DNA/g currants. The antifungal activity of S. cerevisiae Y33 was observed in all studied cases, causing inhibition of fungal growth and OTA production.

Carrier-Mediated Drug Uptake in Fungal Pathogens.

Candida, Aspergillus, and Cryptococcus species are the most frequent cause of severe human fungal infections. Clinically relevant antifungal drugs are scarce, and their effectiveness are hampered by the ability of fungal cells to develop drug resistance mechanisms. Drug effectiveness and drug resistance in human pathogens is very often affected by their "transportome". Many studies have covered a panoply of drug resistance mechanisms that depend on drug efflux pumps belonging to the ATP-Binding Cassette and Major Facilitator Superfamily. However, the study of drug uptake mechanisms has been, to some extent, overlooked in pathogenic fungi. This review focuses on discussing current knowledge on drug uptake systems in fungal pathogens, highlighting the need for further studies on this topic of great importance. The following subjects are covered: (i) drugs imported by known transporter(s) in pathogenic fungi; and (ii) drugs imported by known transporter(s) in the model yeast Saccharomyces cerevisiae or in human parasites, aimed at the identification of their homologs in pathogenic fungi. Besides its contribution to increase the understanding of drug-pathogen interactions, the practical implications of identifying drug importers in human pathogens are discussed, particularly focusing on drug development strategies.

Identification of glucocorticoid receptor in Drosophila melanogaster.

BACKGROUND: Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified. RESULTS: In this study, we demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, we identified the closest genetic GR homolog as D. melanogaster Estrogen Related Receptor (ERR) gene. We discovered that Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene. CONCLUSIONS: This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect.

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.

Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT.

The subcellular localization of proteins is a posttranslational modification of paramount importance. The ability to study subcellular and organelle proteomes improves our understanding of cellular homeostasis and cellular dynamics. In this chapter, we describe a protocol for the unbiased and high-throughput study of protein subcellular localization in the yeast Saccharomyces cerevisiae: hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT), which involves biochemical fractionation of Saccharomyces cerevisiae and high resolution mass spectrometry-based protein quantitation using TMT 10-plex isobaric tags. This protocol enables the determination of the subcellular localizations of thousands of proteins in parallel in a single experiment and thereby deep sampling and high-resolution mapping of the spatial proteome.

Ability of Saccharomyces cerevisiae MC87-46 to assimilate isomaltose and its effects on sake taste.

Recently, wild strains of Saccharomyces cerevisiae isolated from a variety of natural resources have been used to make bread, beer, wine, and sake. In the current study, we isolated wild S. cerevisiae MC strain from the carnation (Dianthus caryophyllus L) flower and produced sake using its cerulenin-resistant mutant strain MC87-46. Then, we characterized the components, including ethanol, amino acids, organic acids, and sugars, in the fermented sake. Sake brewed with MC87-46 is sweet owing to the high content of isomaltose, which was at a concentration of 44.3 mM. The low sake meter value of -19.6 is most likely due to this high isomaltose concentration. The genomic DNA of MC87-46 encodes for isomaltases IMA1, IMA2, IMA3, IMA4 and IMA5, as well as the isomaltose transporter gene, AGT1. However, these genes were not induced in MC87-46 by isomaltose, and the strain did not possess isomaltase activity. These results show that MC87-46 cannot utilize isomaltose, resulting in its accumulation in the fermented sake. Isomaltose concentrations in sake brewed with MC87-46 were 24.6-fold more than in commercial sake. These findings suggest that MC87-46 may be useful for commercial application in Japanese sake production because of its unique flavour and nutrient profile.

Baker's Yeast Clinical Isolates Provide a Model for How Pathogenic Yeasts Adapt to Stress.

Global outbreaks of drug-resistant fungi such as Candida auris are thought to be due at least in part to excessive use of antifungal drugs. Baker's yeast Saccharomyces cerevisiae has gained importance as an emerging opportunistic fungal pathogen that can cause infections in immunocompromised patients. Analyses of over 1000 S. cerevisiae isolates are providing rich resources to better understand how fungi can grow in human environments. A large percentage of clinical S. cerevisiae isolates are heterozygous across many nucleotide sites, and a significant proportion are of mixed ancestry and/or are aneuploid or polyploid. Such features potentially facilitate adaptation to new environments. These observations provide strong impetus for expanding genomic and molecular studies on clinical and wild isolates to understand the prevalence of genetic diversity and instability-generating mechanisms, and how they are selected for and maintained. Such work can also lead to the identification of new targets for antifungal drugs.

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art.

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S. cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.

Filamentation Regulatory Pathways Control Adhesion-Dependent Surface Responses in Yeast.

Signaling pathways can regulate biological responses by the transcriptional regulation of target genes. In yeast, multiple signaling pathways control filamentous growth, a morphogenetic response that occurs in many species including fungal pathogens. Here, we examine the role of signaling pathways that control filamentous growth in regulating adhesion-dependent surface responses, including mat formation and colony patterning. Expression profiling and mutant phenotype analysis showed that the major pathways that regulate filamentous growth [filamentous growth MAPK (fMAPK), RAS, retrograde (RTG), RIM101, RPD3, ELP, SNF1, and PHO85] also regulated mat formation and colony patterning. The chromatin remodeling complex, SAGA, also regulated these responses. We also show that the RAS and RTG pathways coregulated a common set of target genes, and that SAGA regulated target genes known to be controlled by the fMAPK, RAS, and RTG pathways. Analysis of surface growth-specific targets identified genes that respond to low oxygen, high temperature, and desiccation stresses. We also explore the question of why cells make adhesive contacts in colonies. Cell adhesion contacts mediated by the coregulated target and adhesion molecule, Flo11p, deterred entry into colonies by macroscopic predators and impacted colony temperature regulation. The identification of new regulators (e.g., SAGA), and targets of surface growth in yeast may provide insights into fungal pathogenesis in settings where surface growth and adhesion contributes to virulence.

Identification of Novel Components of Target-of-Rapamycin Signaling Pathway by Network-Based Multi-Omics Integrative Analysis.

Target of rapamycin (TOR) is a major signaling pathway and regulator of cell growth. TOR serves as a hub of many signaling routes, and is implicated in the pathophysiology of numerous human diseases, including cancer, diabetes, and neurodegeneration. Therefore, elucidation of unknown components of TOR signaling that could serve as potential biomarkers and drug targets has a great clinical importance. In this study, our aim is to integrate transcriptomics, interactomics, and regulomics data in Saccharomyces cerevisiae using a network-based multiomics approach to enlighten previously unidentified, potential components of TOR signaling. We constructed the TOR-signaling protein interaction network, which was used as a template to search for TOR-mediated rapamycin and caffeine signaling paths. We scored the paths passing from at least one component of TOR Complex 1 or 2 (TORC1/TORC2) using the co-expression levels of the genes in the transcriptome data of the cells grown in the presence of rapamycin or caffeine. The resultant network revealed seven hitherto unannotated proteins, namely, Atg14p, Rim20p, Ret2p, Spt21p, Ylr257wp, Ymr295cp, and Ygr017wp, as potential components of TOR-mediated rapamycin and caffeine signaling in yeast. Among these proteins, we suggest further deciphering of the role of Ylr257wp will be particularly informative in the future because it was the only protein whose removal from the constructed network hindered the signal transduction to the TORC1 effector kinase Npr1p. In conclusion, this study underlines the value of network-based multiomics integrative data analysis in discovering previously unidentified components of the signaling networks by revealing potential components of TOR signaling for future experimental validation.

Acid Stress Triggers Resistance to Acetic Acid-Induced Regulated Cell Death through Hog1 Activation Which Requires RTG2 in Yeast.

Acid stress causes resistance to acetic acid-induced regulated cell death (AA-RCD) in budding yeast, resulting in catalase activation. In order to explore the molecular determinants of evasion of AA-RCD triggered by acid stress adaptation, we studied the involvement and the possible interplay of the master regulator of transcription high-osmolarity glycerol 1 (HOG1) and RTG2, a positive regulator of the RTG-dependent mitochondrial retrograde signaling. Viability, DNA fragmentation, and ROS accumulation have been analyzed in wild-type and mutant cells lacking HOG1 and/or RTG2. Catalase activity and transcription of CTT1 and CTA1, coding the cytosolic and peroxisomal/mitochondrial catalase, respectively, as well as Hog1 phosphorylation, were also analyzed. Our results show that HOG1 is essential for resistance to AA-RCD and its activation results in the upregulation of CTT1, but not CTA1, transcription during acid stress adaptation. RTG2 is required for Hog1-dependent CTT1 upregulation upon acid stress, despite failure of RTG pathway activation. We give evidence that Rtg2 has a cytoprotective role and can act as a general cell stress sensor independent of Rtg1/3-dependent transcription.

Protective role of Syzygium cumini leaf extracts against paraquat-induced oxidative stress in superoxide-dismutase-deficient Saccharomyces cerevisiae strains

The aim of this study was to evaluate the protective effect of three different extracts prepared from Syzygium cumini leaves against paraquat-induced toxicity in Saccharomyces cerevisiaestrains deficient in superoxide dismutase (SOD). Additionally, the extracts phenolic and flavonoid contents, in vitro antioxidant activity, and phytochemical composition (using high-pressure liquid chromatography) were determined. Bioactive compounds from S. cumini leaves were extracted with infusion (traditional method) or ultrasound (aqueous or hydroalcoholic). Compared to the infusion extract, the ultrasound extracts exhibited a greater protective capacity against paraquat toxicity in the yeast cells as well as higher antioxidant activity. These results may be directly related to the higher phenolic and flavonoid contents in these extracts, since they are recognized as having high antioxidant actions.