A novel Hsp90 phospho-switch modulates virulence in the major human fungal pathogen Candida albicans

The ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. Hsp90 also regulates cellular morphogenesis, drug resistance, and virulence in human pathogenic fungi, which kill more than 1.6 million patients each year worldwide. Invasive fungal infections are difficult to treat due to the lack of effective antifungal therapies, resulting in mortality rates of up to 95%. As a key regulator of fungal virulence, Hsp90 is an attractive therapeutic target. However, fungal and animal homologs are highly conserved, impeding fungal-specific targeting. Thus, understanding the factors that regulate Hsp90 could provide an alternative strategy aimed at exclusively targeting this regulator of fungal virulence. Here, we demonstrate how CK2-mediated phosphorylation of two Hsp90 residues modulates virulence in a major fungal pathogen of humans, Candida albicans. We combined proteomics, molecular evolution and structural modelling with molecular biology to identify and characterize two Hsp90 phosphorylation sites. Phosphorylation negatively affects thermal stress response, morphogenesis, drug susceptibility and fungal virulence. Our results provide the first record of specific Hsp90 phosphorylation sites acting as modulators of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitation as antifungal drug targets.

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Transcriptome analysis uncovers a link between copper metabolism, and both fungal fitness and antifungal sensitivity in the opportunistic yeast Candida albicans

10.1101/2020.03.27.011775 ◽

2020 ◽

Author(s):

Inès Khemiri ◽

Faiza Tebbji ◽

Adnane Sellam

Keyword(s):

Candida Albicans ◽

Stress Responses ◽

Copper Metabolism ◽

Pathogenic Fungi ◽

Global Gene Expression ◽

Host Cells ◽

Fungal Virulence ◽

Copper Transporter ◽

Gene Expression Dynamic ◽

As Stress

AbstractCopper homeostasis is an important determinant for virulence of many human pathogenic fungi such as the highly prevalent yeast Candida albicans. However, beyond the copper transporter Ctr1, little is known regarding other genes and biological processes that are affected by copper. To gain insight into the cellular processes that are modulated by copper abundance in C. albicans, we monitored the global gene expression dynamic under both copper depletion and excess using RNA-seq. Beyond copper metabolism, other different transcriptional programs related to fungal fitness such as stress responses, antifungal sensitivity, host invasion and commensalism were modulated in response to copper variations. We have also investigated the transcriptome of the mutant of the copper utilization regulator, mac1, and identified potential direct targets of this transcription factor under copper starvation. We also showed that Mac1 was required for the invasion and adhesion to host cells and antifungal tolerance. This study provides a framework for future studies to examine the link between copper metabolism and essential functions that modulate fungal virulence and fitness inside the host.

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Identification and Phenotypic Characterization of Hsp90 Phosphorylation Sites That Modulate Virulence Traits in the Major Human Fungal Pathogen Candida albicans

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.637836 ◽

2021 ◽

Vol 11 ◽

Author(s):

Leenah Alaalm ◽

Julia L. Crunden ◽

Mark Butcher ◽

Ulrike Obst ◽

Ryann Whealy ◽

...

Keyword(s):

Candida Albicans ◽

Environmental Stress ◽

Stress Responses ◽

Fungal Pathogens ◽

Phosphorylation Site ◽

Pathogenic Fungi ◽

Phenotypic Characterization ◽

Fungal Virulence ◽

Post Translational Modifications ◽

Virulence Traits

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.

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Histone acetylation/deacetylation in Candida albicans and their potential as antifungal targets

Future Microbiology ◽

10.2217/fmb-2019-0343 ◽

2020 ◽

Vol 15 (11) ◽

pp. 1075-1090

Author(s):

Shan Su ◽

Xiuyun Li ◽

Xinmei Yang ◽

Yiman Li ◽

Xueqi Chen ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Histone Acetylation ◽

Stress Responses ◽

Histone Deacetylases ◽

Fungal Pathogen ◽

Antifungal Agents ◽

Fungal Infections ◽

Histone Acetyltransferases ◽

Opportunistic Fungal Pathogen

Recently, the incidence of invasive fungal infections has significantly increased. Candida albicans (C. albicans) is the most common opportunistic fungal pathogen that infects humans. The limited number of available antifungal agents and the emergence of drug resistance pose difficulties to treatment, thus new antifungals are urgently needed. Through their functions in DNA replication, DNA repair and transcription, histone acetyltransferases (HATs) and histone deacetylases (HDACs) perform essential functions relating to growth, virulence, drug resistance and stress responses of C. albicans. Here, we summarize the physiological and pathological functions of HATs/HDACs, potential antifungal targets and underlying antifungal compounds that impact histone acetylation and deacetylation. We anticipate this review will stimulate the identification of new HAT/HDAC-related antifungal targets and antifungal agents.

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In Fungal Intracellular Pathogenesis, Form Determines Fate

mBio ◽

10.1128/mbio.02092-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 4

Author(s):

Robin C. May ◽

Arturo Casadevall

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Membrane Integrity ◽

Pathogenic Fungi ◽

Morphological Transition ◽

Physical Damage ◽

Fungal Cell ◽

Morphological Transitions ◽

Pathogenic Microbes ◽

Phagosomal Membrane

ABSTRACT For pathogenic microbes to survive ingestion by macrophages, they must subvert powerful microbicidal mechanisms within the phagolysosome. After ingestion, Candida albicans undergoes a morphological transition producing hyphae, while the surrounding phagosome exhibits a loss of phagosomal acidity. However, how these two events are related has remained enigmatic. Now Westman et al. (mBio 9:e01226-18, 2018, https://doi.org/10.1128/mBio.01226-18) report that phagosomal neutralization results from disruption of phagosomal membrane integrity by the enlarging hyphae, directly implicating the morphological transition in physical damage that promotes intracellular survival. The C. albicans intracellular strategy shows parallels with another fungal pathogen, Cryptococcus neoformans, where a morphological changed involving capsular enlargement intracellularly is associated with loss of membrane integrity and death of the host cell. These similarities among distantly related pathogenic fungi suggest that morphological transitions that are common in fungi directly affect the outcome of the fungal cell-macrophage interaction. For this class of organisms, form determines fate in the intracellular environment.

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ANTI-CANDIDA ALBICANS ACTIVITY OF THE ASSOCIATION OF CITRONELAL WITH ANFOTERICIN B OR WITH CETOCONAZOLE

Periódico Tchê Química ◽

10.52571/ptq.v16.n31.2019.256_periodico31_pgs_250_257.pdf ◽

2019 ◽

Vol 16 (31) ◽

pp. 250-257

Author(s):

Patrícia Duarte Costa SILVA ◽

Brenda Lavínia Calixto dos SANTOS ◽

Gustavo Lima SOARES ◽

Wylly Araújo de OLIVEIRA

Keyword(s):

Candida Albicans ◽

Antifungal Activity ◽

Amphotericin B ◽

Inhibitory Concentration ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Mortality Rates ◽

New Drugs ◽

High Morbidity ◽

Isolated Species

Fungal infections caused by species of the genus Candida are responsible for high morbidity and mortality rates, mainly affecting immunocompromised individuals. Among fungi, Candida albicans is the most frequently isolated species of clinical specimens. A problem associated with increased resistance of pathogenic fungi to the agents used in the therapeutic regimen and the limited number of drugs to cure these infections. As a result, the search for new drugs with antifungal activity has become increasingly important. The aim of this study is to study the antifungal activity of citronellal alone and in combination with amphotericin B or ketoconazole. The Minimal Inhibitory Concentration of citronellal, amphotericin B and ketoconazole against strains of Candida albicans were evaluated by the microdilution technique, and the Minimum Fungicide Concentration of citronellal against the same strains was also performed. Through the checkerboard methodology the effect of the combination of citronelal with amphotericin B or with ketoconazole was determined. This study showed that the association of citronellal with ketoconazole was shown to be an additive against one of the strains of C. albicans and indifferent to another strain. While the combined activity of citronellal and amphotericin B demonstrated an indifferent effect on the strains tested.

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Cross kingdom analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets to ameliorate fungal pathogenicity?

10.1101/2020.09.23.310581 ◽

2020 ◽

Author(s):

Emily F. Warner ◽

Natália Bohálová ◽

Václav Brázda ◽

Zoë A. E. Waller ◽

Stefan Bidula

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Protein Modification ◽

Pathogenic Fungi ◽

Significant Heterogeneity ◽

Nucleic Acid Binding ◽

Fungal Virulence ◽

Coding Regions ◽

Critical Requirement ◽

Novel Drug

AbstractFungi contribute to upwards of 1.5 million human deaths annually, are involved in the spoilage of up to a third of food crops, and have a devastating effect on plant and animal biodiversity. Moreover, this already significant issue is exacerbated by a rise in antifungal resistance and a critical requirement for novel drug targets. Quadruplexes are four-stranded secondary structures in nucleic acids which can regulate processes such as transcription, translation, replication, and recombination. They are also found in genes linked to virulence in microbes, and quadruplex-binding ligands have been demonstrated to eliminate drug resistant pathogens. Using a computational approach, we identified putative quadruplex-forming sequences (PQS) in 1362 genomes across the fungal kingdom and explored their potential involvement in virulence, drug resistance, and pathogenicity. Here we present the largest analysis of PQS in fungi and identified significant heterogeneity of these sequences throughout phyla, genera, and species. Moreover, PQS were genetically conserved. Notably, loss of PQS in cryptococci and aspergilli was associated with pathogenicity. PQS in the clinically important pathogens Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans were located within genes (particularly coding regions), mRNA, repeat regions, mobile elements, tRNA, ncRNA, rRNA, and the centromere. Genes containing PQS in these organisms were found to be primarily associated with metabolism, nucleic acid binding, transporter activity, and protein modification. Finally, PQS were found in over 100 genes associated with virulence, drug resistance, or key biological processes in these pathogenic fungi and were found in genes which were highly upregulated during germination, hypoxia, oxidative stress, iron limitation, and in biofilms. Taken together, quadruplexes in fungi could present interesting novel targets to ameliorate fungal virulence and overcome drug resistance.

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Fungal Extracellular Vesicles as Potential Targets for Immune Interventions

mSphere ◽

10.1128/msphere.00747-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 5

Author(s):

Mateus Silveira Freitas ◽

Vânia Luiza Deperon Bonato ◽

Andre Moreira Pessoni ◽

Marcio L. Rodrigues ◽

Arturo Casadevall ◽

...

Keyword(s):

Immune System ◽

Immune Responses ◽

Nucleic Acids ◽

Extracellular Vesicles ◽

Fungal Pathogen ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Therapeutic Strategies ◽

Host Immune System ◽

Immunomodulatory Properties

ABSTRACT The release of extracellular vesicles (EVs) by fungi is a fundamental cellular process. EVs carry several biomolecules, including pigments, proteins, enzymes, lipids, nucleic acids, and carbohydrates, and are involved in physiological and pathological processes. EVs may play a pivotal role in the establishment of fungal infections, as they can interact with the host immune system to elicit multiple outcomes. It has been observed that, depending on the fungal pathogen, EVs can exacerbate or attenuate fungal infections. The study of the interaction between fungal EVs and the host immune system and understanding of the mechanisms that regulate those interactions might be useful for the development of new adjuvants as well as the improvement of protective immune responses against infectious or noninfectious diseases. In this review, we describe the immunomodulatory properties of EVs produced by pathogenic fungi and discuss their potential as adjuvants for prophylactic or therapeutic strategies.

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Mitochondrial Cochaperone Mge1 Is Involved in Regulating Susceptibility to Fluconazole in Saccharomyces cerevisiae and Candida Species

mBio ◽

10.1128/mbio.00201-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 14

Author(s):

Liesbeth Demuyser ◽

Erwin Swinnen ◽

Alessandro Fiori ◽

Beatriz Herrera-Malaver ◽

Kevin Verstrepen ◽

...

Keyword(s):

Candida Albicans ◽

Iron Metabolism ◽

Candida Glabrata ◽

Human Population ◽

Bacterial Infections ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Recurrent Infections ◽

Iron Sulfur Clusters ◽

Iron Sulfur

ABSTRACT MGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae. We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicans. IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker’s yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans. IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker’s yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans.

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Structure-guided approaches to targeting stress responses in human fungal pathogens

Journal of Biological Chemistry ◽

10.1074/jbc.rev120.013731 ◽

2020 ◽

Vol 295 (42) ◽

pp. 14458-14472

Author(s):

Emmanuelle V. LeBlanc ◽

Elizabeth J. Polvi ◽

Amanda O. Veri ◽

Gilbert G. Privé ◽

Leah E. Cowen

Keyword(s):

Stress Response ◽

Stress Responses ◽

Fungal Pathogens ◽

Fungal Infections ◽

Fungal Growth ◽

Small Gtpase ◽

Ecological Niches ◽

Attractive Alternative ◽

Response Regulators ◽

Fungal Virulence

Fungi inhabit extraordinarily diverse ecological niches, including the human body. Invasive fungal infections have a devastating impact on human health worldwide, killing ∼1.5 million individuals annually. The majority of these deaths are attributable to species of Candida, Cryptococcus, and Aspergillus. Treating fungal infections is challenging, in part due to the emergence of resistance to our limited arsenal of antifungal agents, necessitating the development of novel therapeutic options. Whereas conventional antifungal strategies target proteins or cellular components essential for fungal growth, an attractive alternative strategy involves targeting proteins that regulate fungal virulence or antifungal drug resistance, such as regulators of fungal stress responses. Stress response networks enable fungi to adapt, grow, and cause disease in humans and include regulators that are highly conserved across eukaryotes as well as those that are fungal-specific. This review highlights recent developments in elucidating crystal structures of fungal stress response regulators and emphasizes how this knowledge can guide the design of fungal-selective inhibitors. We focus on the progress that has been made with highly conserved regulators, including the molecular chaperone Hsp90, the protein phosphatase calcineurin, and the small GTPase Ras1, as well as with divergent stress response regulators, including the cell wall kinase Yck2 and trehalose synthases. Exploring structures of these important fungal stress regulators will accelerate the design of selective antifungals that can be deployed to combat life-threatening fungal diseases.

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