Bigger Data Approach to Analysis of Essential Oils and Their Antifungal Activity against Aspergillus niger, Candida albicans, and Cryptococcus neoformans.
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ABSTRACT:
SUBMITTER: Powers CN
PROVIDER: S-EPMC6718987 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Some species of fungi can cause serious human diseases, particularly to immuno-compromised individuals. Opportunistic fungal infections are a leading cause of mortality, and present an emerging challenge that requires development of new and effective therapeutics. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of cellular protein translation machinery and can be targeted for discovery of novel anti-fungal agents. RESULTS: Validation of aaRSs as potential drug targets in pathogenic microbes prompted us to investigate the genomic distribution of aaRSs within three fungi that infect humans - A. niger, C. albicans and C. neoformans. Hidden Markov Models were built for aaRSs and related proteins to search for homologues in these fungal genomes. Here, we provide a detailed and comprehensive annotation for 3 fungal genome aaRSs and their associated proteins. We delineate predicted localizations, subdomain architectures and prevalence of unusual motifs within these aaRSs. Several fungal aaRSs have unique domain appendages of unknown function e.g. A. niger AsxRS and C. neoformans TyrRS have additional domains that are absent from human homologs. CONCLUSIONS: Detailed comparisons of fungal aaRSs with human homologs suggest key differences that could be exploited for specific drug targeting. Our cataloging and structural analyses provide a comprehensive foundation for experimentally dissecting fungal aaRSs that may enable development of new anti-fungal agents.
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Project description:Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans. In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.
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Project description:Fungal populations are commonly found in natural environments and present enormous health care challenges, due to increased resistance to antifungal agents. Paeonol exhibits antifungal activities; nevertheless, the antifungal and antibiofilm activities of paeonol against Candida albicans and Cryptococcus neoformans remain largely unexplored. Here, we aimed to evaluate the antifungal and antibiofilm activities of paeonol against C. albicans and/or C. neoformans (i.e., against mono- or dual-species). The minimum inhibitory concentrations (MICs) of paeonol for mono-species comprising C. albicans or C. neoformans were 250 μg ml-1, whereas the MIC values of paeonol for dual-species were 500 μg ml-1. Paeonol disrupted cell membrane integrity and increased the influx of gatifloxacin into cells of mono- and dual-species cells, indicating an antifungal mode of action. Moreover, paeonol at 8 times the MIC damaged mono- and dual-species cells within C. albicans and C. neoformans biofilms, as it did planktonic cells. In particular, at 4 and 8 mg ml-1, paeonol efficiently dispersed preformed 48-h biofilms formed by mono- and dual-species cells, respectively. Paeonol inhibited effectively the yeast-to-hyphal-form transition of C. albicans and impaired capsule and melanin production of C. neoformans. The addition of 10 MIC paeonol to the medium did not shorten the lifespan of C. elegans, and 2 MIC paeonol could effectively protect the growth of C. albicans and C. neoformans-infected C. elegans. Furthermore, RNA sequencing was employed to examine the transcript profiling of C. albicans and C. neoformans biofilm cells in response to 1/2 MIC paeonol. RNA sequencing data revealed that paeonol treatment impaired biofilm formation of C. albicans by presumably downregulating the expression level of initial filamentation, adhesion, and growth-related genes, as well as biofilm biosynthesis genes, whereas paeonol inhibited biofilm formation of C. neoformans by presumably upregulating the expression level of ergosterol biosynthesis-related genes. Together, the findings of this study indicate that paeonol can be explored as a candidate antifungal agent for combating serious single and mixed infections caused by C. albicans and C. neoformans.
| S-EPMC9163411 | biostudies-literature
Project description:The increasing prevalence of Candida albicans resistance to commercial antifungal agents in recent decades has prompted modern medicine and veterinary medicine to search for combined treatment options. The aim of the study was to determine the activity of essential oils from different cultivars and morphological parts of the medicinal lavender (Lavandula angustifolia) in combination with fluconazole against Candida albicans ATCC 10231 strain. The effect of the combination of lavender essential oil with fluconazole was tested using the checkerboard method, and the obtained results were interpreted on the basis of fractional inhibitory concentration indices (FICIs). A synergistic interaction was found for all combinations of fluconazole with essential oils isolated both from flowers and leafy stalks of two tested lavender cultivars: 'Blue River' and 'Ellagance Purple'. The observed enhancement effect of fluconazole antifungal activity was significantly stronger in the case of essential oils obtained from flowers and leafy stalks of 'Blue River' cultivar. Analogous studies were performed for linalool, one of the main components of lavender essential oils, and a similar synergistic interaction with fluconazole was found.
| S-EPMC9921790 | biostudies-literature
Project description:The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 μM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals.
| S-EPMC5114273 | biostudies-literature
Project description:Campafungin A is a polyketide that was recognized in the Candida albicans fitness test due to its antiproliferative and antihyphal activity. Its mode of action was hypothesized to involve inhibition of a cAMP-dependent PKA pathway. The originally proposed structure appeared to require a polyketide assembled in a somewhat unusual fashion. However, structural characterization data were never formally published. This background stimulated a reinvestigation in which campafungin A and three closely related minor constituents were purified from fermentations of a strain of the ascomycete fungus Plenodomus enteroleucus. Labeling studies, along with extensive NMR analysis, enabled assignment of a revised structure consistent with conventional polyketide synthetic machinery. The structure elucidation of campafungin A and new analogues encountered in this study, designated here as campafungins B, C, and D, is presented, along with a proposed biosynthetic route. The antimicrobial spectrum was expanded to methicillin-resistant Staphylococcus aureus, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Schizosaccharomyces pombe, with MICs ranging as low as 4-8 μg mL-1 in C. neoformans. Mode-of-action studies employing libraries of C. neoformans mutants indicated that multiple pathways were affected, but mutants in PKA/cAMP pathways were unaffected, indicating that the mode of action was distinct from that observed in C. albicans.
| S-EPMC7530089 | biostudies-literature
Project description:UnlabelledFungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in both Cryptococcus and ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity.ImportanceFungal infections are very common in humans but may be neglected due to misdiagnosis and inattention. Cryptococcus neoformans is a yeast that infects mainly immunocompromised people, causing high mortality rates in developing countries. The fungus infects the lungs, crosses the blood-brain barrier, and invades the cerebrospinal fluid, causing fatal meningitis. C. neoformans infections are treated with amphotericin B, flucytosine, and azoles, all developed decades ago. However, problems with antifungal agents highlight the urgent need for more-effective drugs to treat C. neoformans and other invasive fungal infections. These issues include the negative side effects of amphotericin B, the spontaneous resistance of C. neoformans to azoles, and the inefficacy of the echinocandin antifungals. In this study, we report the identification of C. neoformans essential genes as targets for the development of novel antifungals. Because of the level of evolutionary divergence between C. neoformans and the ascomycetes, a subset of these genes is likely essential in all fungi. Genes identified in this study represent an excellent starting point for the future development of new antifungals by pharmaceutical companies.
| S-EPMC4453551 | biostudies-literature
Project description:Heteroresistance to fluconazole (FLC) in Cryptococcus is a transient adaptive resistance which is lost upon release from the drug pressure. It is known that clones heteroresistant to FLC invariably contain disomic chromosomes, but how disomy is formed remains unclear. Previous reports suggested that the aneuploid heteroresistant colonies in Cryptococcus emerge from multinucleated cells, resembling the case in Candida albicans Although a small number of cells containing multiple nuclei appear in a short time after FLC treatment, we provide evidence that the heteroresistant colonies in the presence of FLC arise from uninucleate cells without involving multinuclear/multimeric stages. We found that fidelity of chromosome segregation in mitosis plays an important role in regulation of FLC heteroresistance frequency in C. neoformans Although FLC-resistant colonies occurred at a very low frequency, we were able to modulate the frequency of heteroresistance by overexpressing SMC1, which encodes a protein containing an SMC domain in chromosome segregation. Using time-lapse microscopy, we captured the entire process of colony formation from a single cell in the presence of FLC. All the multinucleated cells formed within a few hours of FLC exposure failed to multiply after a few cell divisions, and the cells able to proliferate to form colonies were all uninucleate without exception. Furthermore, no nuclear fusion event or asymmetric survival between mother and daughter cells, a hallmark of chromosome nondisjunction in haploid organisms, was observed. Therefore, the mechanisms of aneuploidy formation in C. neoformans appear different from most common categories of aneuploid formation known for yeasts.IMPORTANCE The gold standard of cryptococcosis treatment consists of induction therapy with amphotericin B followed by lifelong maintenance therapy with fluconazole (FLC). However, prolonged exposure to FLC induces the emergence of clones heteroresistant to azoles. All the heteroresistant clones thus far analyzed have been shown to be aneuploids, but how the aneuploid is formed remains unclear. Aneuploidy in fungi and other eukaryotic cells is known to result most commonly from chromosome missegregation during cell division due to defects in any one of the multiple components and processes that are required for the formation of two genetically identical daughter cells. Although formation of multinucleated cells has been observed in cells exposed to FLC, evidence for the emergence of drug-resistant aneuploid populations directly from such cells has been lacking. We show the evidence that the aneuploid in fluconazole-heteroresistant clones of Cryptococcus neoformans is derived neither from multinucleated cells nor from chromosome missegregation.
| S-EPMC6282203 | biostudies-literature
Project description:In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2?/? mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2?/? mutant was unable to grow on a solid medium containing 10 ?g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2?/? mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 ?g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2?/? mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity.
| S-EPMC4135735 | biostudies-literature
Project description:Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.
| S-EPMC1460578 | biostudies-other