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The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOβ2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Fletcher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mediator is a complex of polypeptides that plays a central role in the recruitment of RNA polymerase II to promoters and subsequent transcriptional activation in eukaryotic organisms. Studies have now shown that Mediator has a role in regulating expression of genes implicated in virulence and antifungal drug resistance in pathogenic fungi. The roles of specific Mediator subunits have been investigated in several species of pathogenic fungi, particularly in the most pathogenic yeast Candida albicans. Uniquely, pathogenic yeast also present several interesting examples of divergence in Mediator structure and function, most notably in C. glabrata, which possesses two orthologues of Med15, and in C. albicans, which has a massively expanded family of Med2 orthologues known as the TLO gene family. This review highlights specific examples of recent progress in characterizing the role of Mediator in pathogenic fungi., (© 2023 The Author(s).)

The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature's toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell's ability to cope with environmental stress. Candida albicans , one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections worldwide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modeling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alaalm, Crunden, Butcher, Obst, Whealy, Williamson, O’Brien, Schaffitzel, Ramage, Spencer and Diezmann.)

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. [ABSTRACT FROM AUTHOR]

Candida albicans SC5314 is the most-often used strain for molecular manipulation of the species. The SC5314 reference genome sequence is the result of considerable effort from many scientists and has advanced research into fungal biology and pathogenesis. Although the resource is highly developed and presented in a phased diploid format, the sequence includes gaps and does not extend to the telomeres on its eight chromosome pairs. Accurate SC5314 genome assembly is complicated by the presence of extensive repeated sequences and considerable allelic length variation at some loci. Advances in genome sequencing technology provide the tools to obtain highly accurate long-read data that span even the most-difficult-to-assemble genome regions. Here, we describe derivation of a PacBio HiFi data set and creation of a collapsed haploid telomere-to-telomere assembly of the SC5314 genome (ASM3268872v1) that revealed previously unknown features of the strain. ASM3268872v1 subtelomeric distances were up to 19 kb larger than in the reference genome and revealed a family of highly conserved DNA helicase-encoding genes at 10 of the 16 chromosome ends. We also describe alignments of individual HiFi reads to deduce accurate diploid sequences for the most notoriously difficult-to-assemble C. albicans genes: the agglutinin-like sequence (ALS) gene family. We provide a tutorial that demonstrates how the HiFi reads can be visualized to explore any region of interest. Availability of the HiFi reads data set and the ASM3268872v1 comparative guide assembly will streamline research efforts because accurate diploid sequences can be derived using simple in silico methods rather than time-consuming laboratory-bench approaches. [ABSTRACT FROM AUTHOR]

Introduction: Candida albicans is an opportunistic human pathogen that typically resides as part of the microbiome in the gastrointestinal and genitourinary tracts of a large portion of the human population. This fungus lacks a true sexual cycle and evolves in a largely clonal pattern. The ability to cause disease is consistent across the species as strains causing systemic infections appear across the known C. albicans intra-species clades. Methods: In this work, strains collected from patients with systemic C. albicans infections isolated at the Nebraska Medicine clinical laboratory were typed by MLST analysis. Since the ability to form filaments has been linked to pathogenesis in C. albicans, these clinical strains, as well as a previously genotyped set of clinical strains, were tested for their ability to filament across a variety of inducing conditions. Results: Genotyping of the clinical strains demonstrated that the strains isolated at one of the major medical centers in our region were as diverse as strains collected across the United States. We demonstrated that clinical strains exhibit a variety of filamentation patterns across differing inducing conditions. The only consistent pattern observed in the entire set of clinical strains tested was an almost universal inability to filament in standard solid inducing conditions used throughout the C. albicans field. A different solid filamentation assay that produces more robust filamentation profiles from clinical strains is proposed in this study, although not all strains expected to filament in vivo were filamentous in this assay. Discussion: Our data supports growing evidence that broad phenotypic diversity exists between the C. albicans type strain and clinical strains, suggesting that the type strain poorly represents filamentation patterns observed in most clinical isolates. These data further highlight the need to use diverse clinical strains in pathogenesis assays. [ABSTRACT FROM AUTHOR]

Puccinia spp. causing rust diseases in wheat and other cereals secrete several specialized effector proteins into host cells. Characterization of these proteins and their interaction with host's R proteins could greatly help to limit crop losses due to diseases. Prediction of effector proteins by combining the transcriptome analysis and multiple in-silico approaches is gaining importance in revealing the pathogenic mechanism. The present study involved identification of 13 Puccinia triticina (Pt) coding sequences (CDSs), through transcriptome analysis, that were differentially expressed during wheat-leaf rust interaction; and prediction of their effector like features using different in-silico tools. NCBI-BLAST and pathogenhost interaction BLAST (PHI-BLAST) tools were used to annotate and classify these sequences based on their most closely matched counterpart in both the databases. Homology between CDSs and the annotated sequences in the NCBI database ranged from 79 to 94% and with putative effectors of other plant pathogens in PHI-BLAST from 24.46 to 54.35%. Nine of the 13 CDSs had effector-like features according to EffectorP 3.0 (≥0.546 probability of these sequences to be effector). The qRT-PCR expression analysis revealed that the relative expression of all CDSs in compatible interaction (HD2329) was maximum at 11 days post inoculation (dpi) and that in incompatible interactions (HD2329 + Lr28) was maximum at 3 dpi in seven and 9 dpi in five CDSs. These results suggest that six CDSs (>0.8 effector probability as per EffectorP 3.0) could be considered as putative Pt effectors. The molecular docking and MD simulation analysis of these six CDSs suggested that candidate Lr28 protein binds more strongly to candidate effector c14094_g1_i1 to form more stable complex than the remaining five. Further functional characterization of these six candidate effectors should prove useful for a better understanding of wheat-leaf rust interaction. In turn, this should facilitate effector-based leaf rust resistance breeding in wheat. [ABSTRACT FROM AUTHOR]

Candida sp. are among the most common fungal commensals found in the human microbiome. Although Candida can be found residing harmlessly on the surface of the skin and mucosal membranes, these opportunistic fungi have the potential to cause superficial skin, nail, and mucus membrane infections as well as life threatening systemic infections. Severity of infection is dependent on both fungal and host factors including the immune status of the host. Virulence factors associated with Candida sp. pathogenicity include adhesin proteins, degradative enzymes, phenotypic switching, and morphogenesis. A central transcriptional regulator of morphogenesis, the transcription factor Efg1 was first characterized in Candida albicans in 1997. Since then, EFG1 has been referenced in the Candida literature over three thousand times, with the number of citations growing daily. Arguably one of the most well studied genes in Candida albicans, EFG1 has been referenced in nearly all contexts of Candida biology from the development of novel therapeutics to white opaque switching, hyphae morphology to immunology. In the review that follows we will synthesize the research that has been performed on this extensively studied transcription factor and highlight several important unanswered questions. [ABSTRACT FROM AUTHOR]

Adaptation to acute and chronic stress and/or persistent stressors is a subject of wide interest in central nervous system disorders. In this context, stress is an effector of change in organismal homeostasis and the response is generated when the brain perceives a potential threat. Herein, we discuss a nuanced and granular view whereby a wide variety of genotoxic and environmental stressors, including aging, genetic risk factors, environmental exposures, and age‐ and lifestyle‐related changes, act as direct insults to cellular, as opposed to organismal, homeostasis. These two concepts of how stressors impact the central nervous system are not mutually exclusive. We discuss how maladaptive stressor‐induced changes in protein connectivity through epichaperomes, disease‐associated pathologic scaffolds composed of tightly bound chaperones, co‐chaperones, and other factors, impact intracellular protein functionality altering phenotypes, that in turn disrupt and remodel brain networks ranging from intercellular to brain connectome levels. We provide an evidence‐based view on how these maladaptive changes ranging from stressor to phenotype provide unique precision medicine opportunities for diagnostic and therapeutic development, especially in the context of neurodegenerative disorders including Alzheimer's disease where treatment options are currently limited. [ABSTRACT FROM AUTHOR]

The yeast Candida albicans is primarily a commensal of humans that colonizes the mucosal surfaces of the gastrointestinal and genital tracts. Yet, C. albicans can under certain circumstances undergo a shift from commensalism to pathogenicity. This transition is governed by fungal factors such as morphological transitions, environmental cues for instance relationships with gut microbiota and the host immune system. C. albicans utilizes distinct sets of regulatory programs to colonize or infect its host and to evade the host defense systems. Moreover, an orchestrated iron acquisition mechanism operates to adapt to specific niches with variable iron availability. Studies on regulatory networks and morphogenesis of these two distinct modes of C. albicans growth, suggest that both yeast and hyphal forms exist in both growth patterns and the regulatory circuits are inter‐connected. Here, we summarize current knowledge about C. albicans commensal‐to‐pathogen shift, its regulatory elements and their contribution to human disease. Take aways: Both yeast and hyphal cell co‐exist during commensal and pathogenic growth of C. albicans.Hyphal‐specific virulence factors decide the shift from commensal to pathogenic growth of C. albicans.An integrated regulatory circuit of C. albicans commensalism and pathogenicity evolved to respond to niche‐specific signals. [ABSTRACT FROM AUTHOR]

Candida albicans is a fungal species that can colonize multiple niches in the human host where it can grow either as a commensal or as an opportunistic pathogen. The genome of C. albicans has long been of considerable interest, given that it is highly plastic and can undergo a wide variety of alterations. These changes play a fundamental role in determining C. albicans traits and have been shown to enable adaptation both to the host and to antifungal drugs. C. albicans isolates contain a heterozygous diploid genome that displays variation from the level of single nucleotides to largescale rearrangements and aneuploidy. The heterozygous nature of the genome is now increasingly recognized as being central to C. albicans biology, as the relative fitness of isolates has been shown to correlate with higher levels of overall heterozygosity. Moreover, loss of heterozygosity (LOH) events can arise frequently, either at single polymorphisms or at a chromosomal level, and both can alter the behavior of C. albicans cells during infection or can modulate drug resistance. In this review, we examine genome plasticity in this pathobiont focusing on how gene dosage variation and loss of heterozygosity events can arise and how these modulate C. albicans behavior. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine (GlcNAc) is being increasingly recognized for its ability to stimulate cell signaling. This amino sugar is best known as a component of cell wall peptidoglycan in bacteria, cell wall chitin in fungi and parasites, exoskeletons of arthropods, and the extracellular matrix of animal cells. In addition to these structural roles, GlcNAc is now known to stimulate morphological and stress responses in a wide range of organisms. In fungi, the model organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the ability to respond to GlcNAc or catabolize it, so studies with the human pathogen Candida albicans have been providing new insights into the ability of GlcNAc to stimulate cellular responses. GlcNAc potently induces C. albicans to transition from budding to filamentous hyphal growth. It also promotes an epigenetic switch from White to Opaque cells, which differ in morphology, metabolism, and virulence properties. These studies have led to new discoveries, such as the identification of the first eukaryotic GlcNAc transporter. Other results have shown that GlcNAc can induce signaling in C. albicans in two ways. One is to act as a signaling molecule independent of its catabolism, and the other is that its catabolism can cause the alkalinization of the extracellular environment, which provides an additional stimulus to form hyphae. GlcNAc also induces the expression of virulence genes in the C. albicans, indicating it can influence pathogenesis. Therefore, this review will describe the recent advances in understanding the role of GlcNAc signaling pathways in regulating C. albicans morphogenesis and virulence. [ABSTRACT FROM AUTHOR]

Ginkgo biloba L. has been used in traditional Chinese medicine (TCM) for thousands of years. However, the anti-cancer properties of ginkgolic acids (GAS) isolated from G. biloba have not been investigated in human nasopharyngeal carcinoma cells. In this study, GAS exhibited an inhibitory effect on the ATPase activity of heat shock protein 90 (Hsp90) and anti-proliferative activities against four human cancer cell lines, with IC50 values ranging from 14.91 to 23.81 μg·mL−1. In vivo experiments confirmed that GAS inhibited tumor growth in CNE-2Z cell-xenografted nude mice with low hepatotoxicity. We further demonstrated that GAS suppressed migration and invasion and induced the apoptosis of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (MMP-2, MMP-9, Her-2, c-Raf, Akt, and Bcl-2). Together, GAS are new Hsp90 inhibitors by binding to Hsp90 (hydrogen bond and hydrophobic interaction). Thus, GAS from G. biloba might represent promising Hsp90 inhibitors for the development of anti-nasopharyngeal carcinoma agents. [ABSTRACT FROM AUTHOR]