Opportunistic Human Fungal Pathogen Research Articles

Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts

Plant-pathogenic fungi form intimate interactions with their associated bacterial microbiota during their entire life cycle. However, little is known about the structure, functions and interaction mechanisms of bacterial communities associated with fungal fruiting bodies (perithecia). Here we examined the bacterial microbiome of perithecia formed by Fusarium graminearum, the major pathogenic fungus causing Fusarium head blight in cereals. A total of 111 shared bacterial taxa were identified in the microbiome of 65 perithecium samples collected from 13 geographic locations. Within a representative culture collection, 113 isolates exhibited antagonistic activity against F. graminearum, with Pantoea agglomerans ZJU23 being the most efficient in reducing fungal growth and infectivity. Herbicolin A was identified as the key antifungal compound secreted by ZJU23. Genetic and chemical approaches led to the discovery of its biosynthetic gene cluster. Herbicolin A showed potent in vitro and in planta efficacy towards various fungal pathogens and fungicide-resistant isolates, and exerted a fungus-specific mode of action by directly binding and disrupting ergosterol-containing lipid rafts. Furthermore, herbicolin A exhibited substantially higher activity (between 5- and 141-fold higher) against the human opportunistic fungal pathogens Aspergillus fumigatus and Candida albicans in comparison with the clinically used fungicides amphotericin B and fluconazole. Its mode of action, which is distinct from that of other antifungal drugs, and its efficacy make herbicolin A a promising antifungal drug to combat devastating fungal pathogens, both in agricultural and clinical settings.

Genetic Structure and Triazole Antifungal Susceptibilities of Alternaria alternata from Greenhouses in Kunming, China.

ABSTRACTAlternaria alternata is an opportunistic human fungal pathogen and a ubiquitous phytopathogen capable of causing diseases to >100 agricultural crops and ornamental plants. To control plant diseases caused by A. alternata, triazole fungicides have been widely used both in open crop and vegetable fields and in indoor growth facilities such as greenhouses. At present, the effect of fungicide use on triazole resistance development in A. alternata populations is not known. Here, we isolated 237 A. alternata strains from nine greenhouses around metropolitan Kunming in Yunnan, southwest China, determined their genotypes using 10 short tandem repeat markers, and quantified their susceptibility to four triazoles (difenoconazole, tebuconazole, itraconazole, and voriconazole). Abundant allelic and genotypic diversities were detected among these A. alternata strains. Significantly, over 17% of the strains were resistant to difenoconazole, and both known and new drug-resistance mutations were found in the triazole target gene cyp51. Our findings of high-level genetic variation of A. alternata in greenhouses coupled with high-frequency fungicide resistance call for greater attention to continued monitoring and to developing alternative plant fungal disease management strategies in greenhouses.IMPORTANCE Alternaria alternata is among the most common fungi in our environments, such as indoor facilities, the soil, and outdoor air. It can cause diseases in >100 crop and ornamental plants. Furthermore, it can cause human infections. However, our understanding of its genetic diversity and antifungal susceptibility is very limited. Indeed, the critical threshold values for resistance have not been defined for most antifungal drugs in this species. Greenhouses are known to have heavy applications of agricultural fungicides. In this study, we analyzed strains of A. alternata from nine greenhouses near metropolitan Kunming in southwestern China. Our study revealed very high genetic diversity and identified strains with high MIC values against two agricultural and two medical triazole antifungals within each of the nine greenhouses. Our study calls for greater attention to this emerging threat to food security and human health.

Replicative Aging Remodels the Cell Wall and Is Associated with Increased Intracellular Trafficking in Human Pathogenic Yeasts.

ABSTRACTReplicative aging is an underexplored field of research in medical mycology. Cryptococcus neoformans (Cn) and Candida glabrata (Cg) are dreaded fungal pathogens that cause fatal invasive infections. The fungal cell wall is essential for yeast viability and pathogenesis. In this study, we provide data characterizing age-associated modifications to the cell wall of Cn and Cg. Here, we report that old yeast cells upregulate genes of cell wall biosynthesis, leading to cell wall reorganization and increased levels of all major components, including glucan, chitin, and its derivatives, as well as mannan. This results in a significant thickening of the cell wall in aged cells. Old-generation yeast cells exhibited drastic ultrastructural changes, including the presence of abundant vesicle-like particles in the cytoplasm, and enlarged vacuoles with altered pH homeostasis. Our findings suggest that the cell wall modifications could be enabled by augmented intracellular trafficking. This work furthers our understanding of the cell phenotype that emerges during aging. It highlights differences in these two fungal pathogens and elucidates mechanisms that explain the enhanced resistance of old cells to antifungals and phagocytic attacks.

The yapsin family of aspartyl proteases regulate glucose homeostasis in Candida glabrata

Invasive candidiasis poses a major healthcare threat. The human opportunistic fungal pathogen Candida glabrata, which causes mucosal and deep-seated infections, is armed with distinct virulence attributes, including a family of 11 glycosylphosphatidylinositol-linked aspartyl proteases, CgYapsins. Here, we have profiled total membrane proteomes of the C. glabrata wildtype and 11 proteases-deficient strain, Cgyps1-11Δ, by mass spectrometry analysis and uncovered a novel role for fungal yapsins in glucose sensing and homeostasis. Furthermore, through label-free quantitative membrane proteome analysis, we showed differential abundance of 42% of identified membrane proteins, with electron transport chain and glycolysis proteins displaying lower and higher abundance in Cgyps1-11Δ cells, compared with wildtype cells, respectively. We also demonstrated elevated glucose uptake and upregulation of genes that code for the low-glucose sensor CgSnf3, transcriptional regulators CgMig1 and CgRgt1, and hexose transporter CgHxt2/10 in the Cgyps1-11Δ mutant. We further elucidated a potential underlying mechanism through genetic and transcript measurement analysis under low- and high-glucose conditions and found CgSNF3 deletion to rescue high glucose uptake and attenuated growth of the Cgyps1-11Δ mutant in YPD medium, thereby linking CgYapsins with regulation of the CgSnf3-dependent low-glucose sensing pathway. Last, high ethanol production, diminished mitochondrial membrane potential, and elevated susceptibility to oxidative phosphorylation inhibitors point toward increased fermentative and decreased respiratory metabolism in the Cgyps1-11Δ mutant. Altogether, our findings revealed new possible glucose metabolism-regulatory roles for putative cell surface-associated CgYapsins and advanced our understanding of fungal carbohydrate homeostasis mechanisms.

Role for the phosphatidylinositol 3-phosphate 5-kinase in antifungal tolerance in Candida glabrata

Candida glabrata is an opportunistic fungal pathogen of humans, which is intrinsically less susceptible to widely used azole antifungals, that block ergosterol biosynthesis. The major azole resistance mechanisms include mitochondrial dysfunction and multidrug efflux pump overexpression. In the current study, we have uncovered an essential role for the actin cytoskeletal network reorganization in survival of the azole stress. We demonstrate for the first time that the azole antifungal fluconazole induces remodelling of the actin cytoskeleton in C. glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, we showed that the vacuolar membrane-resident phosphatidylinositol 3-phosphate 5-kinase (CgFab1) is pivotal to this process, as CgFAB1 disruption impaired vacuole homeostasis and actin organization. We also showed that the actin depolymerization factor CgCof1 binds to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and CgCof1 distribution along with the actin filament-capping protein CgCap2 is altered upon both CgFAB1disruption and fluconazole exposure. Additionally, while the F-actin-stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective-mutants, the actin polymerization inhibitor latrunculin B rendered fluconazole fully and partially fungicidal in azole-susceptible and azole-resistant C. glabrata clinical isolates, respectively. These data underscore the essentiality of actin cytoskeleton reorganization for azole stress survival. Lastly, we have also shown a pivotal role of CgFab1 kinase activity regulators, CgFig4, CgVac7 and CgVac14, through genetic analysis, in azole and echinocandin antifungal tolerance. Altogether, I shall present our findings on functions and metabolism of the PI(3,5)P2 lipid in antifungal tolerance and virulence of C. glabrata.

Aspergillus fumigatus pan-genome analysis identifies genetic variants associated with human infection.

Aspergillus fumigatus is an environmental saprobe and opportunistic human fungal pathogen. Despite an estimated annual occurrence of more than 300,000 cases of invasive disease worldwide, a comprehensive survey of the genomic diversity present in A. fumigatus-including the relationship between clinical and environmental isolates and how this genetic diversity contributes to virulence and antifungal drug resistance-has been lacking. In this study we define the pan-genome of A. fumigatus using a collection of 300 globally sampled genomes (83 clinical and 217 environmental isolates). We found that 7,563 of the 10,907 unique orthogroups (69%) are core and present in all isolates and the remaining 3,344 show presence/absence of variation, representing 16-22% of the genome of each isolate. Using this large genomic dataset of environmental and clinical samples, we found an enrichment for clinical isolates in a genetic cluster whose genomes also contain more accessory genes, including genes coding for transmembrane transporters and proteins with iron-binding activity, and genes involved in both carbohydrate and amino-acid metabolism. Finally, we leverage the power of genome-wide association studies to identify genomic variation associated with clinical isolates and triazole resistance as well as characterize genetic variation in known virulence factors. This characterization of the genomic diversity of A. fumigatus allows us to move away from a single reference genome that does not necessarily represent the species as a whole and better understand its pathogenic versatility, ultimately leading to better management of these infections.

Beta vulgaris Assisted Fabrication of Novel Ag-Cu Bimetallic Nanoparticles for Growth Inhibition and Virulence in Candida albicans.

Beta vulgaris extract contains water-soluble red pigment betanin and is used as a food colorant. In this study, the biogenic Ag-Cu bimetallic nanoparticles were synthesized and characterized by different spectroscopic and microscopic techniques, including UV–Visible, FTIR, TEM. SEM-EDX, XRD, and TGA. Further, Ag-Cu bimetallic nanoparticles capped with Beta vulgaris biomolecules were evaluated for their antifungal activity against Candida albicans via targeting its major virulence factors, including adherence, yeast to hyphae transition, extracellular enzyme secretion, biofilm formation, and the expression of genes related to these pathogenic traits by using standard methods. C. albicans is an opportunistic human fungal pathogen that causes significant morbidity and mortality, mainly in immunocompromised patients. The current antifungal therapy is limited with various shortcomings such as host toxicity and developing multidrug resistance. Therefore, the development of novel antifungal agents is urgently required. Furthermore, NPs were screened for cell viability and cytotoxicity effect. Antifungal susceptibility testing showed potent antifungal activity of the Ag-Cu bimetallic NPs with a significant inhibitory effect on adherence, yeast to hyphae transition, extracellular enzymes secretion, and formation of biofilms in C. albicans at sub-inhibitory and inhibitory concentrations. The RT-qPCR results at an MIC value of the NPs exhibited a varying degree of downregulation in expression levels of virulence genes. Results also revealed the dose-dependent effect of NPs on cellular viability (up to 100%) using MUSE cell analyzer. Moreover, the low cytotoxicity effect of bimetallic NPs has been observed using haemolytic assay. The overall results indicated that the newly synthesized Ag-Cu bimetallic NPs capped with Beta vulgaris are proven to possess a potent anticandidal activity, by affecting the vital pathogenic factors of C. albicans.

Set1-mediated H3K4 methylation is required for Candida albicans virulence by regulating intracellular level of reactive oxygen species

ABSTRACT Candida albicans is an opportunistic human fungal pathogen that exists in normal flora but can cause infection in immunocompromised individuals. The transition to pathogenic C. albicans requires a change of various gene expressions. Because histone-modifying enzymes can regulate gene expression, they are thought to control the virulence of C. albicans. Indeed, the absence of H3 lysine 4 (H3K4) methyltransferase Set1 has been shown to reduce the virulence of C. albicans; however, Set1-regulated genes responsible for this attenuated virulence phenotype remain unknown. Here, we demonstrated that Set1 positively regulates the expression of mitochondrial protein genes by methylating H3K4. In particular, levels of cellular mitochondrial reactive oxygen species (ROS) were higher in Δset1 than in the wild-type due to the defect of those genes’ expression. Set1 deletion also increases H2O2 sensitivity and prevents proper colony formation when interacting with macrophage in vitro, consistent with its attenuated virulence in vivo. Together, these findings suggest that Set1 is required to regulate proper cellular ROS production by positively regulating the expression of mitochondrial protein genes and subsequently sustaining mitochondrial membrane integrity. Consequently, C. albicans maintains proper ROS levels via Set1-mediated transcriptional regulation, thus establishing a rapid defense against external ROS generated by the host.

Phenotypic Plasticity in the Productions of Virulence Factors Within and Among Serotypes in the Cryptococcus neoformans Species Complex.

The Cryptococcus neoformans species complex (CNSC) is a common opportunistic human fungal pathogen and the most frequent cause of fungal meningitis. There are three major serotypes in CNSC: A, D, and their hybrids AD, and they have different geographic distributions and medical significance. Melanin pigment and a polysaccharide capsule are the two major virulence factors in CNSC. However, the relationships between serotype and virulence factor production and how environmental factors might impact their relationships are not known. This study investigated the expressions of melanin and capsular polysaccharide in a genetically diverse group of CNSC strains and how their phenotypic expressions were influenced by oxidative and nitrosative stress levels. We found significant differences in melanin and capsular polysaccharide productions among serotypes and across stress conditions. Under oxidative stress, the laboratory hybrids exhibited the highest phenotypic plasticity for melanin production while serotype A showed the highest for capsular polysaccharide production. In contrast, serotype D exhibited the highest phenotypic plasticity for capsular polysaccharide production and clinical serotype AD the highest phenotypic plasticity for melanin production under nitrosative stress. These results demonstrated that different serotypes have different environmental condition-specific mechanisms to modulate the expression of virulence factors.

Anticandidal Potential of Two Cyanobacteria-Synthesized Silver Nanoparticles: Effects on Growth, Cell Morphology, and Key Virulence Attributes of Candida albicans.

Candida albicans is an opportunistic human fungal pathogen responsible for 90–100% of mucosal and nosocomial infections worldwide. The emergence of drug-resistant strains has resulted in adverse consequences for human health, including numerous deaths. Consequently, there is an urgent need to identify and develop new antimicrobial drugs to counter these effects. Antimicrobial nanoagents have shown potent inhibitory activity against a number of pathogens through targeting their defense systems, such as biofilm formation. Here, we investigated the anticandidal activity of silver nanoparticles biosynthesized by the cyanobacterial strains Desertifilum sp. IPPAS B-1220 and Nostoc Bahar_M (D-SNPs and N-SNPs, respectively), along with that of silver nitrate (AgNO3), and examined the mechanisms underlying their lethal effects. For this, we performed agar well diffusion and enzyme activity assays (lactate dehydrogenase, adenosine triphosphatase, glutathione peroxidase, and catalase) and undertook morphological examinations using transmission electron microscopy. The effects of the three treatments on Hwp1 and CDR1 gene expression and protein patterns were assessed using qRT-PCR and SDS–PAGE assays, respectively. All of the three treatments inhibited C. albicans growth; disrupted membrane integrity, metabolic function, and antioxidant activity; induced ultrastructural changes in the cell envelope; and disrupted cytoplasmic and nuclear contents. Of the three agents, D-SNPs showed the greatest biocidal activity against C. albicans. Additionally, the D-SNP treatment significantly reduced the gene expression of Hwp1 and CDR1, suggestive of negative effects on biofilm formation ability and resistance potential of C. albicans, and promoted protein degradation. The mechanism involved in the biocidal effects of both D-SNPs and N-SNPs against C. albicans could be attributed to their ability to interfere with fungal cell structures and/or stimulate oxidative stress, enabling them to be used as a robust antimycotic agent.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

Retinoids in Fungal Infections: From Bench to Bedside.

Retinoids—a class of chemical compounds derived from vitamin A or chemically related to it—are used especially in dermatology, oncohematology and infectious diseases. It has been shown that retinoids—from their first generation—exert a potent antimicrobial activity against a wide range of pathogens, including bacteria, fungi and viruses. In this review, we summarize current evidence on retinoids’ efficacy as antifungal agents. Studies were identified by searching electronic databases (MEDLINE, EMBASE, PubMed, Cochrane, Trials.gov) and reference lists of respective articles from 1946 to today. Only articles published in the English language were included. A total of thirty-nine articles were found according to the criteria. In this regard, to date, In vitro and In vivo studies have demonstrated the efficacy of retinoids against a broad-spectrum of human opportunistic fungal pathogens, including yeast fungi that normally colonize the skin and mucosal surfaces of humans such as Candida spp., Rhodotorula mucilaginosa and Malassezia furfur, as well as environmental moulds such as Aspergillus spp., Fonsecae monofora and many species of dermatophytes associated with fungal infections both in humans and animals. Notwithstanding a lack of double-blind clinical trials, the efficacy, tolerability and safety profile of retinoids have been demonstrated against localized and systemic fungal infections.

Development of Carbazole Derivatives Compounds against Candida albicans: Candidates to Prevent Hyphal Formation via the Ras1-MAPK Pathway.

Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is a transcriptional repressor for the expression of hyphal-specific genes in morphogenesis. Azoles or echinocandin drugs have been extensively prescribed for C. albicans infections, which has led to the development of drug-resistant strains. Therefore, it is necessary to develop new molecules to effectively treat fungal infections. Here, we showed that Molecule B and Molecule C, which contained a carbazole structure, attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that Molecule B and Molecule C inhibit morphogenesis through repressing protein and RNA levels of Ras/MAPK-related genes, including UME6 and NRG1. Furthermore, we determined the antifungal effects of Molecule B and Molecule C in vivo using a candidiasis murine model. We anticipate our findings are that Molecule B and Molecule C, which inhibits the Ras1/MAPK pathway, are promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases.

Aspergillus fumigatus Acetate Utilization Impacts Virulence Traits and Pathogenicity.

ABSTRACTAspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies.

Biodirected Synthesis of Silver Nanoparticles Using Aqueous Honey Solutions and Evaluation of Their Antifungal Activity against Pathogenic Candida Spp.

Silver nanoparticles (AgNPs) were synthesized using aqueous honey solutions with a concentration of 2%, 10%, and 20%—AgNPs-H2, AgNPs-H10, and AgNPs-H20. The reaction was conducted at 35 °C and 70 °C. Additionally, nanoparticles obtained with the citrate method (AgNPs-C), while amphotericin B (AmB) and fluconazole were used as controls. The presence and physicochemical properties of AgNPs was affirmed by analyzing the sample with ultraviolet–visible (UV–Vis) and fluorescence spectroscopy, scanning electron microscopy (SEM), and dynamic light scattering (DLS). The 20% honey solution caused an inhibition of the synthesis of nanoparticles at 35 °C. The antifungal activity of the AgNPs was evaluated using opportunistic human fungal pathogens Candida albicans and Candida parapsilosis. The antifungal effect was determined by the minimum inhibitory concentration (MIC) and disc diffusion assay. The highest activity in the MIC tests was observed in the AgNPs-H2 variant. AgNPs-H10 and AgNPs-H20 showed no activity or even stimulated fungal growth. The results of the Kirby–Bauer disc diffusion susceptibility test for C. parapsilosis strains indicated stronger antifungal activity of AgNPs-H than fluconazole. The study demonstrated that the antifungal activity of AgNPs is closely related to the concentration of honey used for the synthesis thereof.

New cytochalasans from an endophytic Xylaria species associated with Costa Rican Palicourea elata (Rubiaceae)

Four new leucine-derived cytochalasans, possessing a 5,6,5,8-ring (1) and a 5,6,11-ring core (2–4), were isolated from a cultivated endophytic fungus Xylaria sp. strain WH2D4 (Xylariaceae). This fungus was isolated from leaves of the neotropical tree species Palicourea elata (Sw.) Borhidi (Rubiaceae) collected in Costa Rica. The chemical structures were determined by employing IR, MS as well as 1D- and 2D-NMR experiments. The stereochemistry at C-15 of compound 4 was determined by quantum calculations. The isolated compounds did not affect germination and growth of Trichoderma reesei and the opportunistic human fungal pathogen T. longibrachiatum.

Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans

BackgroundCandida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. ResultsHerein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. ConclusionThis analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans.

Repositioning Lopinavir, an HIV Protease Inhibitor, as a Promising Antifungal Drug: Lessons Learned from Candida albicans-In Silico, In Vitro and In Vivo Approaches.

The repurposing strategy was applied herein to evaluate the effects of lopinavir, an aspartic protease inhibitor currently used in the treatment of HIV-infected individuals, on the globally widespread opportunistic human fungal pathogen Candida albicans by using in silico, in vitro and in vivo approaches in order to decipher its targets on fungal cells and its antifungal mechanisms of action. Secreted aspartic proteases (Saps) are the obviously main target of lopinavir. To confirm this hypothesis, molecular docking assays revealed that lopinavir bound to the Sap2 catalytic site of C. albicans as well as inhibited the Sap hydrolytic activity in a typically dose-dependent manner. The inhibition of Saps culminated in the inability of C. albicans yeasts to assimilate the unique nitrogen source (albumin) available in the culture medium, culminating with fungal growth inhibition (IC50 = 39.8 µM). The antifungal action of lopinavir was corroborated by distinct microscopy analyses, which evidenced drastic and irreversible changes in the morphology that justified the fungal death. Furthermore, our results revealed that lopinavir was able to (i) arrest the yeasts-into-hyphae transformation, (ii) disturb the synthesis of neutral lipids, including ergosterol, (iii) modulate the surface-located molecules, such as Saps and mannose-, sialic acid- and N-acetylglucosamine-containing glycoconjugates, (iv) diminish the secretion of hydrolytic enzymes, such as Saps and esterase, (v) negatively influence the biofilm formation on polystyrene surface, (vi) block the in vitro adhesion to epithelial cells, (vii) contain the in vivo infection in both immunocompetent and immunosuppressed mice and (viii) reduce the Sap production by yeasts recovered from kidneys of infected animals. Conclusively, the exposed results highlight that lopinavir may be used as a promising repurposing drug against C. albicans infection as well as may be used as a lead compound for the development of novel antifungal drugs.

Two Functionally Redundant FK506-Binding Proteins Regulate Multidrug Resistance Gene Expression and Govern Azole Antifungal Resistance

Increasing resistance to antifungal therapy is an impediment to the effective treatment of fungal infections. Candida glabrata is an opportunistic human fungal pathogen that is inherently less susceptible to cost-effective azole antifungals. Gain-of-function mutations in the Zn-finger pleiotropic drug resistance transcriptional activator-encoding gene CgPDR1 are the most prevalent causes of azole resistance in clinical settings. CgPDR1 is also transcriptionally activated upon azole exposure; however, factors governing CgPDR1 gene expression are not yet fully understood. Here, we have uncovered a novel role for two FK506-binding proteins, CgFpr3 and CgFpr4, in the regulation of the CgPDR1 regulon. We show that CgFpr3 and CgFpr4 possess a peptidyl-prolyl isomerase domain and act redundantly to control CgPDR1 expression, as a Cgfpr3Δ4Δ mutant displayed elevated expression of the CgPDR1 gene along with overexpression of its target genes, CgCDR1, CgCDR2, and CgSNQ2, which code for ATP-binding cassette multidrug transporters. Furthermore, CgFpr3 and CgFpr4 are required for the maintenance of histone H3 and H4 protein levels, and fluconazole exposure leads to elevated H3 and H4 protein levels. Consistent with the role of histone proteins in azole resistance, disruption of genes coding for the histone demethylase CgRph1 and the histone H3K36-specific methyltransferase CgSet2 leads to increased and decreased susceptibility to fluconazole, respectively, with the Cgrph1Δ mutant displaying significantly lower basal expression levels of the CgPDR1 and CgCDR1 genes. These data underscore a hitherto unknown role of histone methylation in modulating the most common azole antifungal resistance mechanism. Altogether, our findings establish a link between CgFpr-mediated histone homeostasis and CgPDR1 gene expression and implicate CgFpr in the virulence of C. glabrata.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.