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

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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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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Anticapsular and Antifungal Activity of α-Cyperone

Antibiotics ◽

10.3390/antibiotics10010051 ◽

2021 ◽

Vol 10 (1) ◽

pp. 51

Author(s):

Connor Horn ◽

Govindsamy Vediyappan

Keyword(s):

Antifungal Activity ◽

Fungal Pathogens ◽

Fungal Infections ◽

Antifungal Drugs ◽

Cyperus Rotundus ◽

Drug Resistant ◽

Fungal Virulence ◽

Medical Needs ◽

Number Of Patients ◽

Immunosuppressed Patients

Fungal infections affect 300 million people and cause 1.5 million deaths globally per year. With the number of immunosuppressed patients increasing steadily, there is an increasing number of patients infected with opportunistic fungal infections such as infections caused by the species of Candida and Cryptococcus. In fact, the drug-resistant Can. krusei and the emerging pan-antifungal resistant Can. auris pose a serious threat to human health as the existing limited antifungals are futile. To further complicate therapy, fungi produce capsules and spores that are resistant to most antifungal drugs/host defenses. Novel antifungal drugs are urgently needed to fill unmet medical needs. From screening a collection of medicinal plant sources for antifungal activity, we have identified an active fraction from the rhizome of Cyperus rotundus, the nut grass plant. The fraction contained α-Cyperone, an essential oil that showed fungicidal activity against different species of Candida. Interestingly, the minimal inhibitory concentration of α-Cyperone was reduced 8-fold when combined with a clinical antifungal drug, fluconazole, indicating its antifungal synergistic potential and could be useful for combination therapy. Furthermore, α-Cyperone affected the synthesis of the capsule in Cryp. neoformans, a causative agent of fungal meningitis in humans. Further work on mechanistic understanding of α-Cyperone against fungal virulence could help develop a novel antifungal agent for drug-resistant fungal pathogens.

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The Development of a Phytopathogenic Fungi Control Trial: Aspergillus flavus and Aspergillus niger Infection in Jojoba Tissue Culture as a Model

The Scientific World JOURNAL ◽

10.1155/2021/6639850 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Nawal Abd El-Baky ◽

Raoufa Ahmed Abdel Rahman ◽

Mona Mohammed Sharaf ◽

Amro Abd Al Fattah Amara

Keyword(s):

Tissue Culture ◽

Experimental Design ◽

Aspergillus Niger ◽

Aspergillus Flavus ◽

Fungal Pathogens ◽

Fungal Infections ◽

Control Plant ◽

Fungal Growth ◽

Ghost Cells

After introducing the idea of using concentrations equal to or less than the minimum inhibition concentration (MIC) of some active chemical compounds for evacuating microbial cells, different types of microbes were evacuated. The original protocol was given the name sponge-like protocol and then was reduced and modified from a microorganism to another to prepare microbial ghosts for various applications such as immunological applications, drug delivery, and isolation of DNA and protein. Fungal pathogens that infect plants critically affect cost effectiveness, quality, and quantity of their production. They kill plant cells and/or cause plant stress. Plant fungal infections can originate from many sources such as infected soil, seeds, or crop debris causing diseases and quality losses around the world with billions of US dollars annually as costs of the associated productivity loss. This study focused on the application of the sponge-like protocol in protecting in vitro tissue cultures of plants against fungal pathogens. This can be useful for research purposes or may be developed to be introduced in field applications. Aspergillus flavus and Aspergillus niger infection in tissue culture of jojoba (Simmondsia chinensis (Link) Schn.) was used as a model to establish the employment of this protocol to control plant fungal diseases. The best conditions for A. flavus and A. niger ghosts production previously mapped by randomization experimental design (reduced Plackett–Burman experimental design) were used to prepare fungal ghosts. SDS, NaOH, NaHCO3, and H2O2 were used in their MIC (+1 level) or minimum growth concentration (MGC, −1 level) according to the determined optimal experimental design. The release of both of DNA and protein from the fungal cells was evaluated spectrophotometrically at 260nm and 280nm, respectively, as an indicator for cell loss of their cytoplasm. Fungal ghost cells were also examined by transmission electron microscopy. After confirming the preparation of high-quality fungal ghost cells, the same conditions were mimicked to control plant fungal infection. Jojoba grown in tissue culture was sprayed with fungal cells (about 103 CFU) as a control experiment or fungal cells followed by treatment with solution (a) represents the fungal ghost cells formation calculated critical concentration (FGCCC) of SDS, NaOH, and NaHCO3 and then treatment with solution (b) represents H2O2 FGCCC. The plant was examined on day 0 (plant grown before any infection or infection followed by treatment), day 5 (plant at day 5 after infection or infection followed by treatment), and day 10 (plant at day 10 after infection or infection followed by treatment). We observed fungal growth in case of control experiments at days 5 and 10 on the tissue culture medium, as well as plant, and the absence of any fungal growth in case of plant treated with FGCCC even after day 10. We recommend using this FGCCC in the form of chemical spraying formulation to treat the plants aiming to control different plant fungal infections in in vitro tissue culture systems or applied in field.

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UvWHI2 is Required for Stress Response and Pathogenicity in Ustilaginonidea Virens

10.21203/rs.3.rs-101297/v1 ◽

2020 ◽

Author(s):

Shuai Meng ◽

Jiehua Qiu ◽

Meng Xiong ◽

Zhiquan Liu ◽

Jane Sadhna Jagernath ◽

...

Keyword(s):

Stress Response ◽

Stress Responses ◽

Fungal Growth ◽

Growth Stress ◽

Phenotypic Analysis ◽

Ustilaginoidea Virens ◽

General Stress Response ◽

Relative Expression Level ◽

False Smut ◽

Limited Surface

Abstract Background: Ustilaginoidea virens causes rice false smut disease, which emerges as a worldwide disease of rice. At present, some stress response related genes have been identified in U. virens , but it is not clear whether and how defects of stress responses affect the pathogenesis processes of U. virens . To answer this question, the function of a general stress response factor Whi2 was analyzed in U. virens . Results: In this study, we identified UvWhi2 as a homolog of Saccharomyces cerevisiae Whi2 in U. virens. The relative expression level of UvWhi2 was significantly up-regulated during infection, suggesting that UvWhi2 may be involved in pathogenesis. Furthermore, knockout of UvWhi2 showed decreased the mycelial growth, increased in conidiation in the PS (potato sucrose) medium and a defect in pathogenicity. In addition, the RNA-Seq and phenotypic analysis showed that UvWHI2 is involved in response to oxidative, hyperosmotic, cell wall stresses, and nutrient limitation. Further studies revealed that the defects of stress responses of the ∆ Uvwhi2 mutant affected the formation of secondary spores on the nutrient limited surface and the rice surface, resulting in a significant reduction of pathogenicity of U. virens . Conclusions: Our results suggest that UvWhi2 is necessary for fungal growth, stress responses, and the formation of secondary spores in U. virens . In addition, the defects of stress responses could affect the formation of secondary spores on the rice surface, and then compromise the pathogenicity of U. virens .

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Recent Advances in Azole Based Scaffolds as Anticandidal Agents

Letters in Drug Design & Discovery ◽

10.2174/1570180815666180917125916 ◽

2019 ◽

Vol 16 (5) ◽

pp. 492-501 ◽

Cited By ~ 1

Author(s):

Prabhuodeyara Math Gurubasavaraj ◽

Jasmith Shivayya Charantimath

Keyword(s):

Drug Delivery ◽

Drug Resistance ◽

Candida Albicans ◽

Drug Interactions ◽

Fungal Pathogens ◽

Antifungal Agents ◽

Fungal Infections ◽

Antifungal Drugs ◽

Fungal Virulence ◽

Compromised Patients

Aim:The present review aims to explore the development of novel antifungal agents, such as pharmacology, pharmacokinetics, spectrum of activity, safety, toxicity and other aspects that involve drug-drug interactions of the azole antifungal agents.Introduction:Fungal infections in critically ill and immune-compromised patients are increasing at alarming rates, caused mainly by Candida albicans an opportunistic fungus. Despite antifungal annihilators like amphotericin B, azoles and caspofungin, these infections are enormously increasing. The unconventional increase in such patients is a challenging task for the management of antifungal infections especially Candidiasis. Moreover, problem of toxicity associated with antifungal drugs on hosts and rise of drug-resistance in primary and opportunistic fungal pathogens has obstructed the success of antifungal therapy.Conclusion:Hence, to conflict these problems new antifungal agents with advanced efficacy, new formulations of drug delivery and novel compounds which can interact with fungal virulence are developed and used to treat antifungal infections.

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

10.1101/2020.09.23.309831 ◽

2020 ◽

Author(s):

Leenah Alaalm ◽

Julia L. Crunden ◽

Mark Butcher ◽

Ulrike Obst ◽

Ryann Whealy ◽

...

Keyword(s):

Candida Albicans ◽

Stress Responses ◽

Fungal Pathogen ◽

Drug Targets ◽

Fungal Infections ◽

Pathogenic Fungi ◽

First Record ◽

Drug Susceptibility ◽

Phosphorylation Sites ◽

Fungal Virulence

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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Genome-Wide Identification of bZIP Transcription Factor Genes and Functional Analyses of Two Members in Cytospora chrysosperma

Journal of Fungi ◽

10.3390/jof8010034 ◽

2021 ◽

Vol 8 (1) ◽

pp. 34

Author(s):

Dasen Wen ◽

Lu Yu ◽

Dianguang Xiong ◽

Chengming Tian

Keyword(s):

Transcription Factor ◽

Stress Responses ◽

Abiotic Stresses ◽

Pathogenic Fungus ◽

Fungal Growth ◽

Nutrient Utilization ◽

Bzip Transcription Factor ◽

Deletion Mutants ◽

Fungal Virulence ◽

Cytospora Chrysosperma

The basic leucine zipper (bZIP) transcription factor (TF) family, one of the largest and the most diverse TF families, is widely distributed across the eukaryotes. It has been described that the bZIP TFs play diverse roles in development, nutrient utilization, and various stress responses in fungi. However, little is known of the bZIP members in Cytospora chrysosperma, a notorious plant pathogenic fungus, which causes canker disease on over 80 woody plant species. In this study, 26 bZIP genes were systematically identified in the genome of C. chrysosperma, and two of them (named CcbZIP05 and CcbZIP23) significantly down-regulated in CcPmk1 deletion mutant (a pathogenicity-related mitogen-activated protein kinase) were selected for further analysis. Deletion of CcbZIP05 or CcbZIP23 displayed a dramatic reduction in fungal growth but showed increased hypha branching and resistance to cell wall inhibitors and abiotic stresses. The CcbZIP05 deletion mutants but not CcbZIP23 deletion mutants were more sensitive to the hydrogen peroxide compared to the wild-type and complemented strains. Additionally, the CcbZIP23 deletion mutants produced few pycnidia but more pigment. Remarkably, both CcbZIP05 and CcbZIP23 deletion mutants were significantly reduced in fungal virulence. Further analysis showed that CcbZIP05 and CcbZIP23 could regulate the expression of putative effector genes and chitin synthesis-related genes. Taken together, our results suggest that CcbZIP05 and CcbZIP23 play important roles in fungal growth, abiotic stresses response, and pathogenicity, which will provide comprehensive information on the CcbZIP genes and lay the foundation for further research on the bZIP members in C. chrysosperma.

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Dual Regulation of Phosphatidylserine Decarboxylase Expression by Envelope Stress Responses

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.665977 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yasmine Hassoun ◽

Julia Bartoli ◽

Astrid Wahl ◽

Julie Pamela Viala ◽

Emmanuelle Bouveret

Keyword(s):

Stress Response ◽

Stress Responses ◽

Response Regulators ◽

Two Component System ◽

Component System ◽

Basal Expression ◽

Gene Coding ◽

Envelope Stress ◽

Two Component ◽

Artificial Activation

Bacteria adapt to versatile environments by modulating gene expression through a set of stress response regulators, alternative Sigma factors, or two-component systems. Among the central processes that must be finely tuned is membrane homeostasis, including synthesis of phospholipids (PL). However, few genetic regulations of this process have been reported. We have previously shown that the gene coding the first step of PL synthesis is regulated by σE and ppGpp, and that the BasRS (PmrAB) two component system controls the expression of the DgkA PL recycling enzyme. The gene coding for phosphatidylserine decarboxylase, the last step in phosphatidylethanolamine synthesis is another gene in the PL synthesis pathway susceptible of stress response regulation. Indeed, psd appears in transcriptome studies of the σE envelope stress Sigma factor and of the CpxAR two component system. Interestingly, this gene is presumably in operon with mscM coding for a miniconductance mechanosensitive channel. In this study, we dissected the promoter region of the psd-mscM operon and studied its regulation by σE and CpxR. By artificial activation of σE and CpxRA stress response pathways, using GFP transcriptional fusion and western-blot analysis of Psd and MscM enzyme production, we showed that the operon is under the control of two distinct promoters. One is activated by σE, the second is activated by CpxRA and also responsible for basal expression of the operon. The fact that the phosphatidylethanolamine synthesis pathway is controlled by envelope stress responses at both its first and last steps might be important for adaptation of the membrane to envelope perturbations.

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In vitro activity of Indian almond (Terminalia catappa) leaf crude extracts against selected dermatophytes

Annals of Tropical Research ◽

10.32945/atr4314.2021 ◽

2021 ◽

pp. 55-66

Author(s):

Roxanne Joy Colendres ◽

Carl Leonard Pradera

Keyword(s):

Fungal Pathogens ◽

Fungal Infections ◽

Fungal Growth ◽

Trichophyton Mentagrophytes ◽

Screening Tests ◽

Malassezia Pachydermatis ◽

Terminalia Catappa ◽

Crude Extracts ◽

Ethanolic Extracts

Fungal infections caused by Trichophyton mentagrophytes, Aspergillus fumigatus , and Malassezia pachydermatis are among the major contributors to multisystemic health problems such as dermatitis, otitis, and respiratory disorders among humans and animals. This study was conducted to determine the in vitro antifungal activity of Terminalia catappa leaf crude aqueous and ethanolic extracts against these fungal pathogens by measuring the zone of inhibition (ZI) using the agar well diffusion technique. Qualitative phytochemical screening tests were also performed to determine bioactive compounds present in the plant extract. Results show that the plant’s crude aqueous (CAE) and ethanolic extracts (CEE) were found to be effective against all test fungi. M. pachydermatis showed susceptibility towards CAE and CEE from T1 (100%), T2 (75%), T3 (50%) and T4 (25%), with the highest mean ZI of 18.33mm and 13.33, respectively. On the other hand, T. mentagrophytes was inhibited by CAE and CEE at T1 (100%), T2 (75%) and T3 (50%) with the highest mean ZI of 9.67mm and 10.33mm, respectively. At the same time, it was observed that A. fumigatus had reactive sensitivity towards CAE and CEE at T1 (100%) and T2 (75%), with the highest mean ZI of 9.33mm and 10.33mm, respectively. Moreover, phytochemical tests showed that the plant’s leaf crude extracts contain alkaloids, saponins, and tannins, which could potentially inhibit fungal growth.

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The Prevalence Onychomycosis in North Western Region of Rajasthan

International Healthcare Research Journal ◽

10.26440/ihrj/01_10/139 ◽

2018 ◽

Vol 1 (10) ◽

pp. 323-329

Author(s):

Ravindra Kumar ◽

Sanju Pannu ◽

Mukesh Kumar ◽

Om Prakash Yadav

Keyword(s):

Fungal Pathogens ◽

Fungal Infections ◽

Fungal Growth ◽

Morphological Identification ◽

Infection Prevalence ◽

Study Region ◽

Nail Diseases ◽

Accurate Treatment ◽

Microbiological Techniques ◽

One Year

NTRODUCTION: Onychomycosis is a fungal infection of nail caused by dermatophytes, yeast and mould. Onchomycosis infection continues to spread worldwide and found to persist everywhere. Onchomycosis infection prevalence is continuously increasing and the possible fungal pathogens are also increasing. All nail diseases are not fungal in origin hence laboratory investigations are needed to differentiate accurately between fungal infections and other conditions.AIM: to find out mycological profile & morphological identification of fungal agent in onchomycosis infection and their prevalence in a part of Rajasthan.MATERIALS AND METHOD: A total 50 samples of nail clipings were investigated in department of microbiology by using 20% KOH and culture tubes of Sabouraud’s Dextrose Agar (SDA) which were mixed with chloramphenicol and cycloheximide.RESULT: A total of 50 samples (18 male and 32 females) were tested in period of one year (January to December 2016). Among them 28(56%) samples showed fungal growth, the predominat fungual pathogen in present study were Tricophyton spp. 12 (42.86%) followed by yeasts 09 (32.14%) then molds 07(25%). Fungi commonly presented in the middle age, between 31-40 years of age, due to trauma at the work site and in women, due to their wet work.CONCLUSION: This study highlighted that Dermatophyte T.Rubrum as the main fungal pathogen which caused onychomycosis in the study region. As there are several fungi which cause the infection so it is necessary to perform culture for appropriate treatment So, it is imperative to diagnose it properly by using microbiological techniques and to treat it properly. For proper management of onychomycosis, diagnosis and accurate treatment play a key role in better outcome.

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