Microfluidic live-cell imaging of Aspergillus fumigatus and Candida albicans hyphal growth treated with AmBisome and Caspofungin.

Knowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungus Candida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicans essential genes, including 1,195 with high "essentiality confidence" scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential in C. albicans were 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogens C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum and highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies.IMPORTANCE Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

Knowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungus Candida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicans essential genes, including 1,195 with high “essentiality confidence” scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential in C. albicans were 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogens C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum and highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies. IMPORTANCE Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

In a previous paper we demonstrated the results of biological screening of Yemeni basidiomycetes. The present study was aimed to investigate the antimicrobial and the antioxidant activity of further basidiomycetes collected in Yemen. Dichloromethane, methanol and aqueous extracts of the fruiting bodies of 25 species were screened in vitro for their antibacterial activities against three Gram-positive bacteria (Staphyloccocus aureus, Bacillus subtilis, Micrococcus flavus) and two Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), against six human fungal pathogens (Candida albicans, Candida krusei, Aspergillus fumigatus, Mucor sp., Microsporum gypseum, Trichophyton mentagrophytes) and against one non human pathogenic fungus (Candida maltosa). The results indicated that 75 extracts exhibited activity against one or more of the bacteria. The methanol extracts of Agaricus cf. bernardii, Agrocybe pediades, Chlorophyllum molybdites, Coriolopsis polyzona, Ganoderma xylonoides, Pycnoporus sanguineus, Trametes lactinea and Trametes cingulata showed activity against all tested bacteria. The highest antibacterial activity was exhibited by methanol extracts from Chlorophyllum molybdites, Ganoderma xylonoides and Trametes cingulata and Agaricus cf. bernardii, Agrocybe pediades, Coriolopsis polyzona, Pycnoporus sanguineus and Trametes lactinea. The methanol extracts of Chlorophyllum molybdites, Ganoderma xylonoides and Pycnoporus sanguineus showed considerable antifungal activities against the tested fungal strains. Strong antioxidative effects employing the DPPH assay were exhibited by methanol extracts from Chlorophyllum molybdites, Ganoderma xylonoides, Hexagonia velutina, Pycnoporus sanguineus, Trametes lactinea and Trametes cingulata. Our previous and presented studies about 48 basidiomycetes collected in Yemen provide evidence that basidiomycetes from the Arabic region so far should attract more attention as potential source for new biologically active agents.

Candida albicans, the major fungal pathogen in humans, undergoes morphological conversion from yeasts to filamentous growth forms depending upon various environmental conditions. Here, we have identified a C. albicans gene, namely SSN6, encoding a putative global transcriptional co-repressor that is highly homologous to the Saccharomyces cerevisiae Ssn6. The isolated C. albicans SSN6 complemented the pleiotropic phenotypes of S. cerevisiae ssn6 mutation, and its expression levels declined significantly in response to a strong true hyphal inducer, serum. The mutant lacking C. albicans Ssn6 displayed a stubby pseudohyphal growth pattern, derepressed filament-specific genes in response to elevated temperature 37 degrees C and failed to develop true hyphae, extensive filamentation and virulence. Such morphological defects of ssn6/ssn6 mutant were not rescued by overexpression of Tup1, Cph1 or Efg1. Moreover, epistatic analysis showed that, as far as cell morphology was concerned, Ssn6 was epistatic to Tup1 at the higher temperature but that, at the lower temperature, the ssn6/ssn6 tup1/tup1 double mutant grew in a stubby form of pseudohyphae distinct from the phenotypes of either single mutant. Furthermore, overexpression of SSN6 in C. albicans led to enhanced filamentous growth and attenuated virulence. These findings suggest that Ssn6 may function as an activator as well as a repressor of filamentous growth and be a target for candidacidal drugs, as its excess or deficiency resulted in impaired virulence.

A range of fungicides or antifungals are currently deployed to control fungi in agriculture or medicine, but resistance to current agents is growing so new approaches and molecular targets are urgently needed. Recently, different aminoglycoside antibiotics combined with particular transport inhibitors were found to produce strong, synergistic growth-inhibition of fungi, by synergistically increasing the error rate of mRNA translation. Here, focusing on translation fidelity as a novel target for combinatorial antifungal treatment, we tested the hypothesis that alternative combinations of agents known to affect the availability of functional amino acids would synergistically inhibit growth of major fungal pathogens. We screened 172 novel combinations against three phytopathogens (Rhizoctonia solani, Zymoseptoria tritici, and Botrytis cinerea) and three human pathogens (Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus), showing that 48 combinations inhibited strongly the growth of the pathogens; the growth inhibition effect was significantly greater with the agents combined than by a simple product of their individual effects at the same doses. Of these, 23 combinations were effective against more than one pathogen, including combinations comprising food-and-drug approved compounds, e.g., quinine with bicarbonate, and quinine with hygromycin. These combinations [fractional inhibitory combination (FIC) index ≤0.5] gave up to 100% reduction of fungal growth yield at concentrations of agents which, individually, had negligible effect. No synergy was evident against bacterial, plant or mammalian cells, indicating specificity for fungi. Mode-of-action analyses for quinine + hygromycin indicated that synergistic mistranslation was the antifungal mechanism. That mechanism was not universal as bicarbonate exacerbated quinine action by increasing drug uptake. The study unveils chemical combinations and a target process with potential for control of diverse fungal pathogens, and suggests repurposing possibilities for several current therapeutics.

Loss of Arp1, a putative actin-related protein, triggers filamentous and invasive growth and impairs pathogenicity in Candida albicans

Because of the increase in the number of immunocompromised patients, the incidence of invasive fungal infections is growing, but the treatment efficiency remains unacceptably low. The most potent clinical systemic antifungals (azoles) are the derivatives of two scaffolds: ketoconazole and fluconazole. Being the safest antifungal drugs, they still have shortcomings, mainly because of pharmacokinetics and resistance. Here, we report the successful use of the target fungal enzyme, sterol 14α-demethylase (CYP51), for structure-based design of novel antifungal drug candidates by minor modifications of VNI [( R)- N-(1-(2,4-dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)], an inhibitor of protozoan CYP51 that cures Chagas disease. The synthesis of fungi-oriented VNI derivatives, analysis of their potencies to inhibit CYP51s from two major fungal pathogens ( Aspergillus fumigatus and Candida albicans), microsomal stability, effects in fungal cells, and structural characterization of A. fumigatus CYP51 in complexes with the most potent compound are described, offering a new antifungal drug scaffold and outlining directions for its further optimization.

The pathogenic fungi Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans are an increasing cause of human mortality, especially in immunocompromised populations. During colonization and adaptation to various host environments, these fungi undergo morphogenetic alterations that allow for survival within the host. One key environmental cue driving morphological changes is external temperature. The Hsp90 chaperone protein provides one mechanism to link temperature with the signalling cascades that regulate morphogenesis, fungal development and virulence. Candida albicans is a model system for understanding the connections between morphogenesis and Hsp90. Due to the high degree of conservation in Hsp90, many of the connections in C. albicans may be extrapolated to other fungal pathogens or parasites. Examining the role of Hsp90 during development and morphogenesis in these three major fungal pathogens may provide insight into key aspects of adaptation to the host, leading to additional avenues for therapy.

Background: Opportunistic fungal infections such as Candida albicans and Aspergillus fumigatus are increasing, particularly among immunocompromised patients. The use of synthetic antifungal agents is often limited by side effects, resistance, and high costs, thus alternatives from natural sources are needed. Rice straw (Oryza sativa L.), an agricultural by-product rich in bioactive compounds, has been reported to possess antibacterial activity; however, its potential as an antifungal agent has not been extensively investigated. Objective: This study aimed to evaluate the antifungal potential of rice straw (Oryza sativa L.) extract against the growth of two pathogenic fungi, Candida albicans and Aspergillus fumigatus. Methods: The research was conducted experimentally in a laboratory with a quantitative approach. Extraction of rice straw was carried out using maceration with 96% ethanol, followed by fractionation with n-hexane, ethyl acetate, and n-butanol solvents based on polarity differences. Antifungal activity was tested using the well diffusion method, with inhibition zone measurements as an indicator of effectiveness. Results: The results showed that ethyl acetate and n-butanol fractions exhibited significant antifungal activity against Candida albicans and Aspergillus fumigatus, as evidenced by the largest inhibition zones at a concentration of 7%. The ethyl acetate fraction demonstrated an average inhibition zone of 18.25 mm against Candida albicans and 12.83 mm against Aspergillus fumigatus. Conversely, the n-hexane fraction did not show antifungal activity. Statistical analysis using one-way ANOVA revealed significant differences among concentrations and fraction types. Conclusion: This study concludes that rice straw extract, particularly the ethyl acetate fraction, possesses potential as a natural antifungal agent effective against pathogenic fungi, and may be further developed for traditional plant-based therapy.

Candida albicans, one of the most prevalent human fungal pathogens, causes diverse diseases extending from superficial infections to deadly systemic mycoses. Currently, only three major classes of antifungal drugs are available to treat systemic infections: azoles, polyenes, and echinocandins. Alarmingly, the efficacy of these antifungals against C. albicans is hindered both by basal tolerance toward the drugs and the development of resistance mechanisms such as alterations of the drug's target, modulation of stress responses, and overexpression of efflux pumps. Thus, the need to identify novel antifungal strategies is dire. To address this challenge, we screened 3,049 structurally-diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library against a C. albicans clinical isolate and identified 17 molecules that inhibited C. albicans growth by >80% relative to controls. Among the most potent compounds were CMLD013360, CMLD012661, and CMLD012693, molecules representing two distinct chemical scaffolds, including 3-hydroxyquinolinones and a xanthone natural product. Based on structural insights, CMLD013360, CMLD012661, and CMLD012693 were hypothesized to exert antifungal activity through metal chelation. Follow-up investigations revealed all three compounds exerted antifungal activity against non-albicans Candida, including Candida auris and Candida glabrata, with the xanthone natural product CMLD013360 also displaying activity against the pathogenic mould Aspergillus fumigatus. Media supplementation with metallonutrients, namely ferric or ferrous iron, rescued C. albicans growth, confirming these compounds act as metal chelators. Thus, this work identifies and characterizes two chemical scaffolds that chelate iron to inhibit the growth of the clinically relevant fungal pathogen C. albicansIMPORTANCEThe worldwide incidence of invasive fungal infections is increasing at an alarming rate. Systemic candidiasis caused by the opportunistic pathogen Candida albicans is the most common cause of life-threatening fungal infection. However, due to the limited number of antifungal drug classes available and the rise of antifungal resistance, an urgent need exists for the identification of novel treatments. By screening a compound collection from the Boston University Center for Molecular Discovery (BU-CMD), we identified three compounds representing two distinct chemical scaffolds that displayed activity against C. albicans. Follow-up analyses confirmed these molecules were also active against other pathogenic fungal species including Candida auris and Aspergillus fumigatus. Finally, we determined that these compounds inhibit the growth of C. albicans in culture through iron chelation. Overall, this observation describes two novel chemical scaffolds with antifungal activity against diverse fungal pathogens.

This study was conducted to evaluate the effect of aqueous, ethanolic and ethyl acetate extracts from neem leaves on growth of some human pathogens (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Candida albicans and Microsporum gypseum) in vitro. Different concentrations (5, 10, 15 and 20%) prepared from these extracts inhibited the growth of the test pathogens and the effect gradually increased with concentration. The 20% ethyl acetate extract gave the strongest inhibition compared with the activity obtained by the same concentration of the other extracts. High Performance Liquid Chromatography (HPLC) analysis of ethyl acetate extract showed the presence of a main component (nimonol) which was purified and chemically confirmed by Nuclear Magnetic Resonance (NMR) spectroscopic analysis. The 20% ethyl acetate extract lost a part of its antifungal effect after pooling out the nimonol and this loss in activity was variable on test pathogens. The purified nimonol as a separate compound did not show any antifungal activity when assayed against all the six fungal pathogens.

This study was conducted to evaluate the effect of aqueous, ethanolic and ethyl acetate extracts from neem leaves on growth of some human pathogens (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Candida albicans and Microsporum gypseum) in vitro. Different concentrations (5, 10, 15 and 20%) prepared from these extracts inhibited the growth of the test pathogens and the effect gradually increased with concentration. The 20% ethyl acetate extract gave the strongest inhibition compared with the activity obtained by the same concentration of the other extracts. High Performance Liquid Chromatography (HPLC) analysis of ethyl acetate extract showed the presence of a main component (nimonol) which was purified and chemically confirmed by Nuclear Magnetic Resonance (NMR) spectroscopic analysis. The 20% ethyl acetate extract lost a part of its antifungal effect after pooling out the nimonol and this loss in activity was variable on test pathogens. The purified nimonol as a separate compound did not show any antifungal activity when assayed against all the six fungal pathogens.

BackgroundThe availability of sequence data of human pathogenic fungi generates opportunities to develop Bioinformatics tools and resources for vaccine development towards benefitting at-risk patients.DescriptionWe have developed a fungal adhesin predictor and an immunoinformatics database with predicted adhesins. Based on literature search and domain analysis, we prepared a positive dataset comprising adhesin protein sequences from human fungal pathogens Candida albicans, Candida glabrata, Aspergillus fumigatus, Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Blastomyces dermatitidis, Pneumocystis carinii, Pneumocystis jirovecii and Paracoccidioides brasiliensis. The negative dataset consisted of proteins with high probability to function intracellularly. We have used 3945 compositional properties including frequencies of mono, doublet, triplet, and multiplets of amino acids and hydrophobic properties as input features of protein sequences to Support Vector Machine. Best classifiers were identified through an exhaustive search of 588 parameters and meeting the criteria of best Mathews Correlation Coefficient and lowest coefficient of variation among the 3 fold cross validation datasets. The "FungalRV adhesin predictor" was built on three models whose average Mathews Correlation Coefficient was in the range 0.89-0.90 and its coefficient of variation across three fold cross validation datasets in the range 1.2% - 2.74% at threshold score of 0. We obtained an overall MCC value of 0.8702 considering all 8 pathogens, namely, C. albicans, C. glabrata, A. fumigatus, B. dermatitidis, C. immitis, C. posadasii, H. capsulatum and P. brasiliensis thus showing high sensitivity and specificity at a threshold of 0.511. In case of P. brasiliensis the algorithm achieved a sensitivity of 66.67%. A total of 307 fungal adhesins and adhesin like proteins were predicted from the entire proteomes of eight human pathogenic fungal species. The immunoinformatics analysis data on these proteins were organized for easy user interface analysis. A Web interface was developed for analysis by users. The predicted adhesin sequences were processed through 18 immunoinformatics algorithms and these data have been organized into MySQL backend. A user friendly interface has been developed for experimental researchers for retrieving information from the database.ConclusionFungalRV webserver facilitating the discovery process for novel human pathogenic fungal adhesin vaccine has been developed.

Candida albicans, a major human fungal pathogen, can form biofilms on a variety of inert and biological surfaces. C. albicans biofilms allow for immune evasion, are highly resistant to antifungal therapies, and represent a significant complication for a wide variety of immunocompromised patients in clinical settings. While transcriptional regulators and global transcriptional profiles of C. albicans biofilm formation have been well-characterized, much less is known about translational regulation of this important C. albicans virulence property. Here, using ribosome profiling, we define the first global translational profile of genes that are expressed during early biofilm development in a human fungal pathogen, C. albicans. We show that C. albicans biofilm formation involves altered translational regulation of genes and gene classes associated with protein synthesis, pathogenesis, transport, plasma membrane, polarized growth, cell cycle, secretion, and signal transduction. Interestingly, while similar, but not identical, classes of genes showed transcriptional alterations during early C. albicans biofilm development, we observed very little overlap between specific genes that are upregulated or downregulated at the translational vs transcriptional levels. Our results suggest that distinct translational mechanisms play an important role in regulating early biofilm development of a major human fungal pathogen. These mechanisms, in turn, could serve as potential targets for novel antifungal strategies.IMPORTANCEThe major human fungal pathogen Candida albicans is known to form biofilms or complex aggregated microbial communities encased in an extracellular matrix. These biofilms allow C. albicans to escape detection by the immune system as well as resist a variety of antifungal drugs. In this study, we define the first global profile of genes that show altered translation during C. albicans biofilm formation. These genes are involved in a variety of key cellular processes, including polarized growth, pathogenesis, transport, protein synthesis, cell cycle, plasma membrane, signal transduction, and secretion. Interestingly, while similar classes of genes are induced at both the transcriptional and translational levels during early C. albicans biofilm formation, we observed very little overlap among specific genes with altered transcription and translation. Our results suggest that C. albicans biofilm formation is controlled by distinct translational mechanisms, which could potentially be targeted by novel antifungal drugs.

Candida albicans has been known to be a major opportunistic fungal pathogen in human, especially threatening life of immunocompromised patients. The polymorphic cell type allows C. albicans to adapt to different environmental conditions and to colonize different tissues in hosts. Morphological transition of C. albicans is closely associated with cell cycle. SCF complexes are key regulators via ubiquitin-proteasome system in cell cycle control. The SCF complexes function as E3 ubiquitin ligase and consist of common components Skp1p, Cul1p and a variable component of F-box protein such as Cdc4p for substrate recognition. SCFCdc4p plays an important role for ubiquitination of specific target proteins to enter S phase from G1 in Saccharomyces cerevisiae. S. cerevisiae CDC4 (ScCDC4) is an essential gene, mutation of which gives rise to cell cycle arrest at G1/S transition. We have identified a homologue of ScCDC4 in C. albicans (CaCDC4) by its ability to rescue the temperature sensitivity of S. cerevisiae cdc4-3 mutant. However, C. albicans grows as filaments in the absence of CaCDC4, suggesting that CaCdc4p serve as negative regulation of filamentation and its influence about cell cycle is unknown. I have been interested in understanding the dramatic functional change between CaCDC4 and ScCDC4. Accordingly, to determine the domain function of CaCdc4p for morphogenesis, I have carried out dissecting the function of CaCdc4p domains in C. albicans with the Tet-on system, the pTET25M and its derivatives, which I have made improvement from the original pTET25 for broader application. Under the Tet-on system, the doxycycline-inducible expression of assorted CaCdc4p domains detected by Western blotting revealed the instability nature of CaCdc4p due to its cleavage at N-terminal region and weak signal of full-length CaCdc4p, suggesting that regulation of stability of CaCdc4p may be important for its function. By expressing each of assorted CaCdc4p domains under the Tet-on system in a C. albicans strain with one CaCDC4 allele deleted and the other under control of CaMET3 promoter simultaneously, JSCA0021 and its derivatives, the phenotypic consequences containing morphological alteration of cell form in the liquid medium and colonies on the agar solely from each of those domains were assessed under microscope and stereo microscope. It appeared that domains of F-box and WD40 repeats, known to responsible for interaction with Skp1p of SCF complex and substrate specificity, respectively, were critical for the function of CaCdc4p, because phenotypes in strains with or without expression of each of these two domains under the CaMET3 promoter repressed condition were the same into filamentous growth and cell aggregation (flocculation). Interestingly, the doxycycline-induced full-length CaCdc4p decreased cell aggregation (flocculation) rather than suppressed filamentous development in the strain where another CaCDC4 being simultaneously repressed by CaMET3 promoter. As a result, I suggested that perhaps time required for the CaMET3 promoter repression is swift than that for the Tet-on induction and that irreversibility of cells from filament to yeast in C. albicans. In addition, I suggested a novel function of CaCDC4 for negatively regulating adhesion as cells lacking CaCDC4 increase the ability of flocculation, which is normally not associated with filamentation. Optimization of the phenotypic analysis and development of quantitative assays are thus crucial to adequately examine the domain function of CaCdc4p.