Ergosterol Biosynthesis Inhibitors Research Articles

Oryzapsins, the orthologs of yeast yapsin in Aspergillus oryzae, affect ergosterol synthesis.

The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.Key points• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.• The oryzapsins would be involved in ergosterol biosynthesis.

The Medicinal Chemistry of 3-nitro-1,2,4-triazoles: Focus on Infectious Diseases.

Infectious diseases are among the leading causes of death worldwide, especially in developing countries. The historical lack of interest of the pharmaceutical industry in developing new drugs against many of these diseases, such as tuberculosis, leishmaniasis, Chagas disease, sleeping sickness, and fungal infections, has left millions of individuals dependent on old treatments that are often ineffective and present different adverse effects. In this sense, new substances against these diseases must be identified. A class of substances that has stood out in the search for new drugs against these diseases is azole derivatives. Within this class, the 3-nitro-1,2,4-triazole nucleus has attracted increasing interest due to its potential, specifically when compared to the 1,2,4-triazole nucleus without the presence of the nitro group, and also in relation to the 2-nitroimidazole nucleus, showing greater potency and selectivity against different etiological agents. This is even more relevant considering that 3-nitro-1,2,4-triazolic substances can promote their activity through different mechanisms of action, such as the inhibition of ergosterol biosynthesis and also via activation by the nitroreductase enzyme, which can avoid the development of cross-resistance. Therefore, in this review, the medicinal chemistry of nitrotriazoles is discussed through the analysis of their potential in terms of biological activity against the etiological agents of several diseases, such as Chagas disease, sleeping sickness and leishmaniasis, caused by kinetoplastid parasites, tuberculosis, caused by the mycobacteria Mycobacterium tuberculosis, and against different species of pathogenic fungi. In addition, aspects related to enzymatic activities, molecular modeling and organic synthesis of these substances are also addressed.

Chitosan nanoencapsulation of Pogostemon cablin (Blanco) Benth. essential oil and its novel preservative effect for enhanced shelf life of stored Maize kernels during storage: Evaluation of its enhanced antifungal, antimycotoxin, antioxidant activities and possible mode of action

AbstractThe present evaluation aimed to synthesis and characterise the Pogostemon cablin essential oil encapsulated chitosan nanoemulsion (PCEO‐CN) and evaluated its antifungal antimycotoxin and antioxidant activities. A total of twenty‐six different chemical compounds were identified from P. cablin essential oil (PCEO), among which patchoulol (34.93%), α‐bulnesene (17.76%) and α‐guaiene (15.44%) were recorded as major components. The average size of PCEO‐CN was 18.20 nm. The PCEO‐CN showed concentration‐dependent broad‐spectrum antifungal and antimycotoxin activities. The in vivo evaluation showed that PCEO‐CN significantly protected maize seeds from mould‐induced biodeterioration and aflatoxin B1 biosynthesis for up to 30 days. The PCEO‐CN significantly inhibits ergosterol biosynthesis and cellular integrities of A. flavus. Based on the present study, PCEO‐CN could be used as a food preservative to control mould and mycotoxin contamination in stored maize.

Antifungal Activities of cis-trans Citral Isomers against Trichophyton rubrum with ERG6 as a Potential Target.

Trichophyton rubrum causes ringworm worldwide. Citral (CIT), extracted from Pectis plants, is a monoterpene and naturally composed of geometric isomers neral (cis-citral) and geranial (trans-citral). CIT has promising antifungal activities and ergosterol biosynthesis inhibition effects against several pathogenic fungi. However, no study has focused on neral and geranial against T. rubrum, which hinders the clinical application of CIT. This study aimed to compare antifungal activities of neral and geranial and preliminarily elucidate their ergosterol biosynthesis inhibition mechanism against T. rubrum. Herein, the disc diffusion assays, cellular leakage measurement, flow cytometry, SEM/TEM observation, sterol quantification, and sterol pattern change analyses were employed. The results showed geranial exhibited larger inhibition zones (p < 0.01 or 0.05), higher cellular leakage rates (p < 0.01), increased conidia with damaged membranes (p < 0.01) within 24 h, more distinct shriveled mycelium in SEM, prominent cellular material leakage, membrane damage, and morphological changes in TEM. Furthermore, geranial possessed more promising ergosterol biosynthesis inhibition effects than neral, and both induced the synthesis of 7-Dehydrodesmosterol and Cholesta-5,7,22,24-tetraen-3β-ol, which represented marker sterols when ERG6 was affected. These results suggest geranial is more potent than neral against T. rubrum, and both inhibit ergosterol biosynthesis by affecting ERG6.

The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure.

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Identification of Thiazoyl Guanidine Derivatives as Novel Antifungal Agents Inhibiting Ergosterol Biosynthesis for Treatment of Invasive Fungal Infections.

Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of existing drugs are unsatisfactory because of side effects, drug-drug interaction, unfavorable pharmacokinetic profiles, and emerging drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure-activity relationship development and property optimization by reducing lipophilicity led to the discovery of 6h, which showed potent antifungal activity against Aspergillus fumigatus in the presence of serum, improved metabolic stability, and PK properties. In the murine systemic A. fumigatus infection model, 6h exhibited antifungal efficacy equivalent to voriconazole (1e). Furthermore, owing to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistant A. fumigatus.

Pantoea vagans strain BWL1 controls blue mold in mandarin fruit by inhibiting ergosterol biosynthesis in Penicillium expansum

Penicillium expansum is a major postharvest fungal pathogen of a variety of fruits. Recently, Pen. expansum has been reported to infect citrus fruit; however, the management of Pen. expansum on citrus has not been studied. Pantoea vagans strain BWL1 was isolated from the surface of mandarin fruit. Here, its inhibitory effect on postharvest blue mold on mandarin fruit was evaluated. Pan. vagans inhibited conidial germination and mycelial growth of Pen. expansum. Cell-free fermentation extract of Pan. vagans inhibited the expression of ergC, a gene involved in ergosterol biosynthesis, and decreased the ergosterol content in Pen. expansum, resulting in plasma membrane disruption in the fungal pathogen. In vivo experiments showed that the development of blue mold caused by Pen. expansum on citrus fruit was significantly decreased by treatment with Pan. vagans strain BWL1. These results suggest that Pan. vagans can produce metabolites that exhibit antifungal activity toward Pen. expansum by inhibiting the biosynthesis of ergosterol. The application of Pan. vagans strain BWL1 could be effective for the postharvest management of blue mold in citrus fruit.

Triadimefon triggers circling behavior and conditioned place preference/aversion in zebrafish in a dose dependent manner

Triadimefon (TDF) is a pesticide used in agricultural crops to control powdery mildews, rusts and other fungal pests. It exerts its fungicidal activity through the inhibition of ergosterol biosynthesis, impairing the formation of the cell membrane. For vertebrates, one of its side effects is the binding to the dopamine transporter increasing the levels of synaptic dopamine, similarly to cocaine. In addition, it has been demonstrated that TDF affects the abundance of other monoamines in the brain, specifically serotonin. It is well known that drugs which alter the dopaminergic and serotonergic systems produce behavioral changes and participate in the development of addictions in mammals. In this work we have used the conditioned place preference paradigm to assess, for the first time, the rewarding properties of TDF in zebrafish. We found out that TDF triggers both, preference and aversion depending on the dosage used during conditioning. We observed that 5 mg/L produced aversion to the pattern previously paired with TDF. However, 15 mg/L induced the opposite behavior, showing that zebrafish seek out those environments which had previously been paired with the higher dose of TDF. These results are congruent with our previous findings, where we showed that 5 mg/L reduced the levels of serotonin, usually linked to anxious behaviors (a negative cue), whereas higher concentrations of TDF increased extracellular dopamine, the main currency of the reward system. Interestingly, both doses of TDF induced circling behavior, a feature usually seen in glutamatergic antagonists.

Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-β-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Combining Miconazole and Domiphen Bromide Results in Excess of Reactive Oxygen Species and Killing of Biofilm Cells.

Fungal biofilm-related infections are increasingly occurring. We previously identified a fungicidal antibiofilm combination, consisting of miconazole (MCZ) and the quaternary ammonium compound domiphen bromide (DB). DB eliminates tolerance rather than altering the susceptibility to MCZ of various Candida spp. Here we studied the mode of action of the MCZ-DB combination in more detail. We found that DB's action increases the permeability of the plasma membrane as well as that of the vacuolar membrane of Candida spp. Furthermore, the addition of DB affects the intracellular azole distribution. MCZ is a fungicidal azole that, apart from its well-known inhibition of ergosterol biosynthesis, also induces accumulation of reactive oxygen species (ROS). Interestingly, the MCZ-DB combination induced significantly more ROS in C. albicans biofilms as compared to single compound treatment. Co-administration of the antioxidant ascorbic acid resulted in abolishment of the ROS generated by MCZ-DB combination as well as its fungicidal action. In conclusion, increased intracellular MCZ availability due to DB's action results in excess of ROS and enhanced fungal cell killing.

Glutamine metabolism modulates azole susceptibility in Trypanosoma cruzi amastigotes.

The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the role of metabolic heterogeneity. We found that limiting exogenous glutamine protects actively dividing amastigotes from ergosterol biosynthesis inhibitors (azoles), independent of parasite growth rate. The antiparasitic properties of azoles are derived from inhibition of lanosterol 14α-demethylase (CYP51) in the endogenous sterol synthesis pathway. We find that carbons from 13C-glutamine feed into amastigote sterols and into metabolic intermediates that accumulate upon CYP51 inhibition. Incorporation of 13C-glutamine into endogenously synthesized sterols is increased with BPTES treatment, an inhibitor of host glutamine metabolism that sensitizes amastigotes to azoles. Similarly, amastigotes are re-sensitized to azoles following addition of metabolites upstream of CYP51, raising the possibility that flux through the sterol synthesis pathway is a determinant of sensitivity to azoles and highlighting the potential role for metabolic heterogeneity in recalcitrant T. cruzi infection.

The Effect of Honokiol on Ergosterol Biosynthesis and Vacuole Function in Candida albicans.

Ergosterol, an essential constituent of membrane lipids of yeast, is distributed in both the cell membrane and intracellular endomembrane components such as vacuoles. Honokiol, a major polyphenol isolated from Magnolia officinalis, has been shown to inhibit the growth of Candida albicans. Here, we assessed the effect of honokiol on ergosterol biosynthesis and vacuole function in C. albicans. Honokiol could decrease the ergosterol content and upregulate the expression of genes related with the ergosterol biosynthesis pathway. The exogenous supply of ergosterol attenuated the toxicity of honokiol against C. albicans. Honokiol treatment could induce cytosolic acidification by blocking the activity of the plasma membrane Pma1p H+-ATPase. Furthermore, honokiol caused abnormalities in vacuole morphology and function. Concomitant ergosterol feeding to some extent restored the vacuolar morphology and the function of acidification in cells treated by honokiol. Honokiol also disrupted the intracellular calcium homeostasis. Amiodarone attenuated the antifungal effects of honokiol against C. albicans, probably due to the activation of the calcineurin signaling pathway which is involved in honokiol tolerance. In conclusion, this study demonstrated that honokiol could inhibit ergosterol biosynthesis and decrease Pma 1p H+-ATPase activity, which resulted in the abnormal pH in vacuole and cytosol.

Fortified Fluconazole Eyedrops for Treatment of Culvularia Corneal Ulcer

Abstract&#x0D; Introduction. Fungal keratitis is less common than bacterial keratitis, generally representing less than 5%–10% of corneal infections in reported clinical series in the United States. But in developing countries (Ghana, India, China) it accounts for more than 90% of the cases. Corneal trauma by plant or vegetative material is the leading risk factor for fungal keratitis. One or more topical antifungals are usually administered with systemic support of oral antifungals. The most common classes of antifungal used for medical therapy include the polyenes, azoles, and the echinocandins.&#x0D; Case Illustration. A 47-year-old male presented with a widening white patch on his left eye since 4 days before coming to our hospital. The left eye was injured by wood flakes 3 weeks ago. He felt a sore, red, watery left eye and blurred vision. He washed his eyes with betel leaf water. His left eye visual acuity was 1/60 and not improved with pinhole. There were blepharospasm, ciliary and conjunctival injection. Corneal examination showed a cloudy cornea, a central corneal defect sized 8x6 mm with an irregular margin, 2/3 to stromal depth, infiltrate, satellite lesion, and positive fluorescein staining at the defect margin. KOH examination demonstrated hyphae. The culture test of left corneal discharge identified a fungi species (Curvularia sp). We prescribed fortified fluconazole eye drops in combination with supportive therapy for 2.5 months. This regimen showed a significant increase in visual acuity to 6/30 with pinhole improvement of 6/21 in the left eye.&#x0D; Discussion. Imidazole acts by inhibiting ergosterol biosynthesis of the fungal cell wall, through action on the cytochrome P450-dependent enzyme. This leads to cell membrane destabilization and leakage. Curvularia sp belongs to the family of dematiaceous fungi. Its clinical manifestations include raised lesions and feathery edges; hypopyon rarely occurs, and the prognosis is quite good. Fluconazole (2 mg/ml) is available for injection and is a well-tolerated eye drop. The intravenous injection can be used as a topical treatment and can be given subconjunctivally at the same concentration.&#x0D; Conclusions. Fortified fluconazole eyedrops (2 mg/ml) can become an alternative and additional therapy for deep fungal keratitis, especially for Curvularia sp.

Biogenic Synthesis of Silver Nanoparticles Using Streptomyces spp. and their Antifungal Activity Against Fusarium verticillioides

The present study reports the synthesis of silver nanoparticles (AgNPs) using haloalkaliphilic Streptomyces spp. characterization, and antifungal activity thereof. The UV visible spectra of synthesized AgNPs showed a characteristic absorption peak at 430 nm, due to the excitation of Surface Plasmon Resonance. Scanning electron microscopy and transmission electron microscopy images showed spherical shape NPs with an average particle size of 16.4 ± 2.2 nm. The crystalline structure of the AgNPs was confirmed by X-ray diffraction (XRD). Zeta potential analyses revealed that NPs were negatively charged (− 8.12 ± 3.87 mV). The synthesized AgNPs are significantly active against phytopathogenic fungi, Fusarium verticillioides and Ustilago maydis. Microscopic, histo- and bio-chemical investigation of AgNPs against F. verticillioides revealed that AgNPs at 100 μg concentration inhibits the hyphal growth and conidia germination, and ~ 42.85% reduction of ergosterol biosynthesis. The results of propidium iodide staining and high relative cell membrane conductivity confirmed AgNPs mediated damage to the membrane. Moreover, the AgNPs synthesized by Streptomyces spp. inhibit the growth of F. verticillioides could be due to the inhibition of ergosterol biosynthesis and membrane damage. In our knowledge, this is the first report demonstrating the anti F. verticillioides activity of AgNPs synthesized by Streptomyces spp.

Molecular fingerprints of conazoles via functional genomic profiling of Saccharomyces cerevisiae

Conazoles were designed to inhibit ergosterol biosynthesis. Conazoles have been widely used as agricultural fungicides and are frequently detected in the environment. Although conazoles have been reported to have adverse effects, such as potential carcinogenic effects, the underlying molecular mechanisms of toxicity remain unclear. Here, the molecular fingerprints of five conazoles (propiconazole (Pro), penconazole (Pen), tebuconazole (Teb), flusilazole (Flu) and epoxiconazole (Epo)) were assessed in Saccharomyces cerevisiae (yeast) via functional genome-wide knockout mutant profiling. A total of 169 (4.49%), 176 (4.67%), 198 (5.26%), 218 (5.79%) and 173 (4.59%) responsive genes were identified at three concentrations (IC50, IC20 and IC10) of Pro, Pen, Teb, Flu and Epo, respectively. The five conazoles tended to have similar gene mutant fingerprints and toxicity mechanisms. “Ribosome” (sce03010) and “cytoplasmic translation” (GO: 0002181) were the common KEGG pathway and GO biological process term by gene set enrichment analysis of the responsive genes, which suggested that conazoles influenced protein synthesis. Conazoles also affected fatty acids synthesis because “biosynthesis of unsaturated fatty acids” pathway was among the top-ranked KEGG pathways. Moreover, two genes, YGR037C (acyl-CoA-binding protein) and YCR034W (fatty acid elongase), were key fingerprints of conazoles because they played vital roles in conazole-induced toxicity. Overall, the fingerprints derived from the yeast functional genomic screening provide an alternative approach to elucidate the molecular mechanisms of environmental pollutant conazoles.

Efficacy of Cinnamomum cassia essential oil against food-borne molds and aflatoxin B1 contamination

The present study deals with the antifungal and antiaflatoxigenic potential of Cinnamomum cassia essential oil (CCEO) against aflatoxigenic food borne Aspergillus flavus strain (AF-LHP-PE-4), and probable mode of action including the safety profile on male mice. CCEO completely inhibited fungal growth and aflatoxin B1 secretion at its minimum inhibitory concentration (0.06 µL/mL). CCEO inhibited ergosterol biosynthesis in cell membranes and also disturbed the membrane fluidity. Significant enhancement in leakage of cellular ions and 260 and 280 nm absorbing cellular materials in response to increased concentrations of CCEO indicated the plasma membrane as the probable site of antifungal toxicity. The antiaflatoxigenic potency of CCEO was confirmed in terms of reduction in the level of cellular methylglyoxal (MG), the inducer of aflatoxin. Antioxidant activity of CCEO was confirmed through DPPH free radical scavenging activity as well as total phenolic content. Chemical profiling of CCEO by gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of cinnamaldehyde (84.01%) as the most abundant compound. CCEO exhibited a high LD50 (10410.75 µL/kg) on male mice, strengthening its favorable safety profile. This is the first report on CCEO as novel green preservative against food storage molds and aflatoxin B1 secretion.

Amlodipine Increases the Therapeutic Potential of Ravuconazole upon Trypanosoma cruzi Infection.

Mining existing agents that enhance the therapeutic potential of ergosterol biosynthesis inhibitors (EBI) is a promising approach to improve Chagas disease chemotherapy. In this study, we evaluated the effect of ravuconazole, an EBI, combined with amlodipine, a calcium channel blocker, upon Trypanosoma cruzi experimental infection. In vitro assays confirmed the trypanocidal activity of both compounds in monotherapy and demonstrated an additive effect (sum of the fractional inhibitory concentration [ΣFIC] > 0.5) of the combined treatment without additional toxicity to host cells. In vivo experiments, using a murine model of the T. cruzi Y strain in a short-term protocol, demonstrated that amlodipine, although lacking trypanocidal activity, dramatically increased the antiparasitic activity of underdosing ravuconazole regimens. Additional analysis using long-term treatment (20 days) showed that parasitemia relapse until 60 days after treatment was significatively lower in mice treated with the combination (4 out of 14 mice) than ravuconazole monotherapy (10 out of 14 mice), even in the presence of immunosuppressant pressure. Furthermore, the combined therapy was well tolerated and protected the mice from mortality. The treatments also impacted on the cellular and humoral immune response of infected animals, inducing a reduction of serum cytokine levels in all ravuconazole-treated mice. Our findings demonstrate that amlodipine is efficacious in enhancing the antiparasitic activity of ravuconazole in an experimental model of T. cruzi infection and indicates a potential strategy to be explored in Chagas disease treatment.

Effect of Posaconazole in an in vitro model of cardiac fibrosis induced by Trypanosoma cruzi

Posaconazole (POS) is an inhibitor of ergosterol biosynthesis in clinical use for treating invasive fungal infections. POS has potent and selective anti-Trypanosoma cruzi activity and has been evaluated as a possible treatment for Chagas disease. Microtissues are a 3D culture system that has been shown to reproduce better tissue architecture and functionality than cell cultures in monolayer (2D). It has been used to evaluate chemotropic response as in vitro disease models. We previously developed an in vitro model that reproduces aspects of cardiac fibrosis observed in Chagas cardiomyopathy, using microtissues formed by primary cardiac cells infected by the T. cruzi, here called T. cruzi fibrotic cardiac microtissue (TCFCM). We also showed that the treatment of TCFCM with a TGF-β pathway inhibitor reduces fibrosis. Here, we aimed to evaluate the effect of POS in TCFCM, observing parasite load and molecules involved in fibrosis. To choose the concentration of POS to be used in TCFCM we first performed experiments in a monolayer of primary cardiac cell cultures and, based on the results, TCFCM was treated with 5 nM of POS for 96 h, starting at 144 h post-infection. Our previous studies showed that at this time the TCFCM had established fibrosis, resulting from T. cruzi infection. Treatment with POS of TCFCM reduced 50 % of parasite load as observed by real-time PCR and reduced markedly the fibrosis as observed by western blot and immunofluorescence, associated with a strong reduction in the expression of fibronectin and laminin (45 % and 54 %, respectively). POS treatment also changed the expression of proteins involved in the regulation of extracellular matrix proteins (TGF-β and TIMP-4, increased by 50 % and decreased by 58 %, respectively) in TCFCM. In conclusion, POS presented a potent trypanocidal effect both in 2D and in TCFCM, and the reduction of the parasite load was associated with a reduction of fibrosis in the absence of external immunological effectors.

Novel structural CYP51 mutation in Trypanosoma cruzi associated with multidrug resistance to CYP51 inhibitors and reduced infectivity

Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, have been proposed as drug candidates for Chagas disease, a neglected infectious tropical disease caused by the protozoan parasite Trypanosoma cruzi. To understand better the mechanism of action and resistance to these inhibitors, a clone of the T. cruzi Y strain was cultured under intermittent and increasing concentrations of ravuconazole until phenotypic stability was achieved. The ravuconazole-selected clone exhibited loss in fitness in vitro when compared to the wild-type parental clone, as observed in reduced invasion capacity and slowed population growth in both mammalian and insect stages of the parasite. In drug activity assays, the resistant clone was above 300-fold more tolerant to ravuconazole than the sensitive parental clone, when the half-maximum effective concentration (EC50) was considered. The resistant clones also showed reduced virulence in vivo, when compared to parental sensitive clones. Cross-resistance to posaconazole and other CYP51 inhibitors, but not to other antichagasic drugs that act independently of CYP51, such as benznidazole and nifurtimox, was also observed. A novel amino acid residue change, T297M, was found in the TcCYP51 gene in the resistant but not in the sensitive clones. The structural effects of the T297M, and of the previously described P355S residue changes, were modelled to understand their impact on interaction with CYP51 inhibitors.

Antifungal activity of cinnamaldehyde against Fusarium sambucinum involves inhibition of ergosterol biosynthesis

In this study, the antifungal effect of cinnamaldehyde against Fusarium sambucinum and its underlying mechanisms were determined. Minimum inhibitory concentration and minimal fungicidal concentration of cinnamaldehyde were 3 and 4mmoll-1 on spore germination and colony development assays in vitro, respectively. Furthermore, the lesion diameter of potato tubers and tuber slices inoculated with F. sambucinum was reduced by 76·9 and 69% after treatment with 4mmoll-1 cinnamaldehyde. Cytometric analyses revelled that cinnamaldehyde significantly affected the integrity of cell membrane firstly, then decreased mitochondrial membrane potential and induced the accumulation of intracellular reactive oxygen species. Meanwhile, high-performance liquid chromatography results indicated that 3mmoll-1 cinnamaldehyde could reduce the ergosterol content by 67·94%. This effect was accompanied by a down-regulation of ERG11, ERG6 and ERG4 which were involved in ergosterol biosynthesis. Theses results suggest that cinnamaldehyde exerts strong antifungal activity against F. sambucinum, probably by affecting the ergosterol biosynthetic processes what leads to the disruption of cell membrane integrity. Cinnamaldehyde is a predominant constituent and key flavour compound of cinnamon essential oil. It has been used as a food additive and flavorant. It is expected to be a novel and safe fungicide for controlling dry rot in potato tubes.