Inhibition Of Ergosterol Synthesis Research Articles

Antisepsis, Disinfection Sterilization - Methods Used in Dentistry

The Pasteur principles regarding the cause of infection and the measures that need to be taken in order to prevent it have become a necessity of the first rank, representing, at the same time, the decisive factor of progress in modern dentistry. Nowadays, based on these principles, from the simplest bandage to the most complex surgery, these are carried out with the help of materials, dental hands and a surgery area where the pathogenic germs have been destroyed. These measures are based on asepsis and antisepsis, methods that perform the disinfection. Asepsis represents the prophylactic side of the disinfection and addresses all the moments that present the risk of contamination. It represents the set of measures by means of which the contact between germs and surgery or accidental plague is avoided. It is a prophylactic method. Antisepsis represents the totality of the means by which the destruction of the germs present in a plague, on the skin or in the environment is sought; it is a curative method. The main mechanisms of action of antiseptics on microorganisms are: altered outer membrane, amino group blockage, tiolitic group blockage, inhibition of ergosterol synthesis, cytoplasmic membrane alteration, energy decoupling, intracytoplasmic clotting. The study comprises 135 sterilizations performed over a period of two years; 107 (79.26%) were done in autoclaves, 7 (5.18%) in ethylene oxide, and 21 (15.56%) in hydrogen peroxide. The use of an antiseptic for long periods of time must be avoided due to the fact that, in the case of those containing heavy metals in their structure, they can be cumulated in the human body, and when toxic concentrations are reached, various toxicities can be triggered.

Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil

Context: Ocimum basilicum L. (Lamiaceae) has been used in folk medicine to treat headaches, kidney disorders, and intestinal worms. Objective: This study evaluates the anti-cryptococcal activity of ethanol crude extract and hexane fraction obtained from O. basilicum var. Maria Bonita leaves. Materials and methods: The MIC values for Cryptococcus sp. were obtained according to Clinical and Laboratory Standards Institute in a range of 0.3–2500 μg/mL. The checkerboard assay evaluated the association of the substances tested (in a range of 0.099–2500 μg/mL) with amphotericin B and O. basilicum essential oil for 48 h. The ethanol extract, hexane fraction and associations in a range of 0.3–2500 μg/mL were tested for pigmentation inhibition after 7 days of treatment. The inhibition of ergosterol synthesis and reduction of capsule size were evaluated after the treatment with ethanol extract (312 μg/mL), hexane fraction (78 μg/mL) and the combinations of essential oil + ethanol extract (78 μg/mL + 19.5 μg/mL, respectively) and essential oil + hexane fraction (39.36 μg/mL + 10 μg/mL, respectively) for 24 and 48 h, respectively. Results: The hexane fraction presented better results than the ethanol extract, with a low MIC (156 μg/mL against C. neoformans T444 and 312 μg/mL against C. neoformans H99 serotype A and C. gattii WM779 serotype C). The combination of the ethanol extract and hexane fraction with amphotericin B and essential oil enhanced their antifungal activity, reducing the concentration of each substance needed to kill 100% of the inoculum. The substances tested were able to reduce the pigmentation, capsule size and ergosterol synthesis, which suggest they have important mechanisms of action. Conclusions: These results provide further support for the use of ethanol extracts of O. basilicum as a potential source of antifungal agents.

Mechanisms of antifungal and anti-aflatoxigenic properties of essential oil derived from turmeric (Curcuma longa L.) on Aspergillus flavus.

The antifungal activity and potential mechanisms in vitro as well as anti-aflatoxigenic efficiency in vivo of natural essential oil (EO) derived from turmeric (Curcuma longa L.) against Aspergillus flavus was intensively investigated. Based on the previous chemical characterization of turmeric EO by gas chromatography-mass spectrometry, the substantially antifungal activities of turmeric EO on the mycelial growth, spore germination and aflatoxin production were observed in a dose-dependent manner. Furthermore, these antifungal effects were related to the disruption of fungal cell endomembrane system including the plasma membrane and mitochondria, specifically i.e. the inhibition of ergosterol synthesis, mitochondrial ATPase, malate dehydrogenase, and succinate dehydrogenase activities. Moreover, the down-regulation profiles of turmeric EO on the relative expression of mycotoxin genes in aflatoxin biosynthetic pathway revealed its anti-aflatoxigenic mechanism. Finally, the suppression effect of fungal contamination in maize indicated that turmeric EO has potential as an eco-friendly antifungal agent.

Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis.

Inhibition of Δ24-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker® Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.

Anti-cryptococcus Activity and Mechanism of Essential Oil from Coreopsis tinctoria Flowering on Cryptococcus

The essential oil (EO) extracted from the flower of Coreopsis tinctoria Nutt. is a kind of plant-derived natural therapeutics against Cryptococcus neoformans. In our study, the anti-cryptococcal effects of the EO were observed from various aspects, such as minimal inhibitory concentration, minimum fungicidal concentration, germ tube formation, and biomass. The results indicated that the EO had potent antifungal activities on C. neoformans and that it was more effective than the commonly used antifungal agents fluconazole and amphotericin B. To further elucidate the mechanism of the antifungal action, the effect of the EO on the plasma membrane and the mitochondria of C. neoformans were investigated. The lesion in the plasma membrane was detected through flow cytometry and was further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of C. neoformans and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential, mitochondrial ATPase, succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) were detected. The reactive oxygen species (ROS) accumulation was examined through fluorometric assay. Decreased SDH, MDH, and ATPase in C. neoformans cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in C. neoformans. A reduction in cell viability was prevented through the addition of L-cysteine, and this reduction indicates that ROS is an important mediator of the antifungal action of the EO. In summary, EO from C. tinctoria flower showed anti-cryptococcus activity. Its mechanism of action is to disrupt the permeability barrier of the plasma membrane and the mitochondrial dysfunction-induced ROS accumulation in C. neoformans. With better antifungal activity than the commonly used antifungal agents and less possibility to induce drug resistance, the EO can be used as a potential anti-cryptococcal agent.

Bio-analytical method development and validation for the estimation of Clotrimazole in human plasma by RP-HPLC method

The present study Estimation of drugs in biological media is increasingly important nowadays, which reveals information like bioavailability, bioequivalence, drug abuse, and pharmacokinetics and drug research. HPLC is the most suitable technique for the analysis of biological fluids owing to its well-developed characteristics and ruggedness. It is an extremely sensitive, precise, accurate, and rapid, separation technique. Clotrimazole is a broad spectrum antifungal agent that is used for the treatment of infections caused by various species of pathogenic dermatophytes, yeasts, and Malassezia furfur. The primary action of Clotrimazole is against dividing and growing organisms. Clotrimazole interacts with yeast 14-α demethylase, a cytochrome P-450 enzyme that converts lanosterol to ergosterol, an essential component of the membrane. In this way, Clotrimazole inhibits ergosterol synthesis, resulting in increased cellular permeability. Few analytical methods were reported for the quantitative determination of Clotrimazole and its combination with other drugs by HPLC. Only one method was reported for Clotrimazole in mice plasma by capillary electrophoresis. But there is no RP-HPLC method reported for the bio-analytical method development in human plasma using protein precipitation method under different chromatographic conditions. The present study is planned to develop newer analytical method for the determination of Clotrimazole by bio-analytical method development in human plasma using protein precipitation by RP-HPLC under different chromatographic conditions. CONCLUSION: A bioanalytical method was developed for the estimation of Clotrimazole by HPLC method. The method was validated for its transferability to other user or other laboratory. The HPLC method was developed by using 0.5%TEA in water (pH 3 adjusted with Orthophosphoric acid) and Acetonitrile in meticulous ratio. The peaks obtained for the drugs of interest by the present method are well resolved from each other without any interference and from the plasma endogenous proteins. The peaks are symmetrical with acceptable tailing factor. The retention time of Clotrimazole was within the limit. The results of linearity, intraday and interday precision study and capability of the extraction method were within the limits of bioanalytical method development. The method was linear with a correlation coefficient of acceptable agreement, which is suitable for the estimation of Clotrimazole in human plasma and other biological fluids. The method demonstrated relative recoveries with acceptable relative standard deviation. The limit of quantification (LOQ) and limit of detection (LOD) for Clotrimazole was found to be nanograms lesser than unity. Hence the developed method is sensitive for the estimation of Clotrimazole in trace amounts. Peak purity studies, with peak purity index values closer to unity reveals that the method developed was specific for the estimation of Clotrimazole in blood and other biological fluids. It can be concluded that the developed RP-HPLC method in human plasma was found to be very simple, reliable and selective for providing satisfactory accuracy and precision. The methods are suitable for routine quantitative analysis in pharmaceutical dosage forms. Hence this developed method can be used further in Bioequivalence and bioavailability studies of clotrimazole. Pharmacokinetic and bio-equivalence study centers

β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine derivatives eradicate C. albicans in an ex vivo human dentinal tubule model by inhibiting sterol 14-α demethylase and cAMP pathway

BackgroundFurther quest for new anti-fungal compounds with proven mechanisms of action arises due to resistance and dose limiting toxicity of existing agents. Among the human fungal pathogens C. albicans predominate by infecting several sites in the body and in particular oral cavity and root canals of human tooth. MethodsIn the present study, we screened a library of β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine compounds against Candida sp. Detailed molecular studies were carried out with the active compound 3 on C. albicans. Morphological damage and antibiofilm activity of compound 3 on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. In silico docking studies were carried out to elucidate the mechanism of action of compound 3. Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model. ResultsScreening data showed that several new compounds were active against Candida sp. Among them, Compound 3 was most potent and exerted time kill effect at 4h, post antifungal effect up to 6h. When used in combination with fluconazole or nystatin, compound 3 revealed an minimum inhibitory concentration (MIC) decrease by 4 fold for both drugs used. In-depth molecular studies with compound 3 on C. albicans showed that this compound inhibited yeast to hyphae (Y-H) conversion and this involved the cAMP pathway. Further, SEM images of C. albicans showed that compound 3 caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, in silico studies revealed that compound 3 docks with the active site of the key enzyme 14-α-demethylase and this might inhibit ergosterol synthesis. In support of this, ergosterol levels were found to be decreased by 32 fold in compound 3 treated samples as analyzed by high performance liquid chromatography (HPLC). Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed 83% eradication of C. albicans and a 6 log reduction in colony forming unit (CFU) after 24h treatment in the infected tooth samples in this model. ConclusionCompound 3 was found to be very effective in eradicating C. albicans by inhibiting cAMP pathway and ergosterol biosynthesis. General SignificanceThe results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.

Interference and Mechanism of Dill Seed Essential Oil and Contribution of Carvone and Limonene in Preventing Sclerotinia Rot of Rapeseed

This study aimed to evaluate the inhibitory effects of dill (Anethum graveolens L.) seed essential oil against Sclerotinia sclerotiorum and its mechanism of action. The antifungal activities of the two main constituents, namely carvone and limonene, were also measured. Mycelial growth and sclerotial germination were thoroughly inhibited by dill seed essential oil at the 1.00 μL/mL under contact condition and 0.125μL/mL air under vapor condition. Carvone also contributed more than limonene in inhibiting the growth of S. sclerotiorum. Carvone and limonene synergistically inhibited the growth of the fungus. In vivo experiments, the essential oil remarkably suppressed S. sclerotiorum, and considerable morphological alterations were observed in the hyphae and sclerotia. Inhibition of ergosterol synthesis, malate dehydrogenase, succinate dehydrogenase activities, and external medium acidification were investigated to elucidate the antifungal mechanism of the essential oil. The seed essential oil of A. graveolens can be extensively used in agriculture for preventing the oilseed crops fungal disease.

Inhibitory effect of nerol against Aspergillus niger on grapes through a membrane lesion mechanism

The antifungal activity of Nerol (NEL) was evaluated against Aspergillus niger, a known cause of grape spoilage, and possible modes of action were explored. The antifungal efficacy of NEL against A. niger has been proven in a dose-dependent manner through in vitro (mycelial growth and spore germination) and in vivo (grapes) tests. An obvious increase in the membrane permeability was detected through the determination of extracellular pH and conductivity. The damage in the cell membrane was measured by monitoring the influx of Propidium iodide (PI) using flow cytometry and was further verified through the inhibition of ergosterol synthesis in the cell membrane. NEL induced morphological changes, and the integrity of the A. niger fungal cells decreased with increasing NEL concentrations. A dose-dependent decrease in ergosterol production was also seen in A. niger cell membranes upon exposure to NEL. The mechanism-of-action study revealed that NEL activated a membrane-active mechanism that inhibits ergosterol synthesis and consequently compromised the membrane integrity of A. niger. As a result, the membrane permeability changes and causes cell death. The membrane lesion mechanism makes NEL a natural alternative to commercial fungicide for control of stored grape spoilage.

Efficacy and possible mechanisms of perillaldehyde in control of Aspergillus niger causing grape decay

A variety of plant products have been recognized for their antifungal activity and recently have attracted food industry attention for their efficacy in controlling postharvest fungal decay of fruits. The antifungal activity of perillaldehyde (PAE) was evaluated against Aspergillus niger, a known cause of grape spoilage, and possible mechanisms were explored. PAE showed notable antifungal activity against A. niger, with a minimum inhibitory concentration (MIC) and a minimum fungicidal concentration (MFC) of 0.25 and 1μl/ml, respectively. The accumulation of mycelial biomass was also inhibited by PAE in a dose-dependent manner, completely inhibiting mycelial growth at 1μl/ml. In vivo data confirmed that the vapour treatment of grapes with various concentrations of PAE markedly improved control of A. niger and suppressed natural decay. Concentrations of PAE of 0.075μl/ml air showed the greatest inhibition of fungal growth compared to the controls. Further experiments indicated that PAE activated a membrane-active mechanism that inhibits ergosterol synthesis, increases membrane permeability (as evidenced by extracellular pH and conductivity measurements), and disrupts membrane integrity, leading to cell death. Our findings suggest that this membrane-active mechanism makes PAE a promising potential antifungal agent for postharvest control of grape spoilage.

A Patient With Ulcerated Nodules on His Face

Diagnosis: Cutaneous squamous cell carcinoma (SCC) likely due to the long-term use of voriconazole. In some clinical settings, ulcerated nodular skin lesions can be concerning for a cutaneous nontuberculosis mycobacterial or fungal infection (Figure 1). However, in our patient, the histological sections of skin from the right mandibular region demonstrated dermal proliferation with nuclear atypia and numerous mitotic figures typical of SCC (Figure 2). In addition, the cells had abundant eosinophilic cytoplasm with intracellular bridging confirming the squamous lineage. The overlying surface epithelium was relatively unremarkable and all fungal and nontuberculosis mycobacterial specific stains and cultures were negative. A computed tomographic (CT) scan of the head and neck revealed periauricular skin thickening compatible with SCC with focal dermal lesions along the face in addition to necrotic lymphadenopathy with apparent invasion into the floor of the mouth. Based on the biopsy and CT findings, the patient was diagnosed with stage IV cutaneous SCC (T4N1M0, with various bilateral cervical lymph nodes). He underwent his initial treatment with local radiotherapy (40 Gy in 16 fractions), which was complicated by hemoptysis. His chronic fungal infection, decreased pulmonary function, and overall poor performance status prohibited the use of systemic myelosuppressive chemotherapy; therefore, a palliative course of cetuximab (epidermal growth factor receptor inhibitor) was administered. Unfortunately, despite these interventions, his SCC progressed over a period of several months and eventually led to airway compromise and death. Voriconazole is a broad-spectrum second-generation azole that exerts its antifungal activity by inhibiting ergosterol synthesis [1]. It has activity against all Candida species, most Aspergillus species, Fusarium species, the endemic mycoses, and Scedosporium apiospermum [1]. Voriconazole is generally well tolerated, but side effects can include self-limiting visual disturbances (photophobia, altered Figure 2. Histologic section from right mandibular region skin biopsy (hematoxylin-eosin stain, ×100 magnification), demonstrating squamous cell carcinoma. The image shows a dermal proliferation with nuclear atypia and numerous mitotic figures (small arrow). The cells have abundant eosinophilic cytoplasm with intracellular bridging confirming the squamous lineage (large arrow). Figure 1. Skin lesions on the right side of the face; diffuse nontender, hard, ulcerated nodules with necrosis that oozed serous fluid.

Preclinical pharmacological profile of Eberconazole: A review and update

Eberconazole is a broad-spectrum imidazole antifungal agent used as a topical preparation in the management of cutaneous mycoses. In vitro studies have shown that eberconazole is effective against dermatophytes, candidiasis, yeasts (including those which are triazole resistant) and Pityriasis versicolor. It inhibits fungal lanosterol 14α-demethylase, thereby inhibiting ergosterol synthesis leading to inhibition of fungal growth. In addition to its antifungal activity, it is also effective against Gram-positive bacteria, a property that is useful clinically. It also possesses anti-inflammatory property thus making it a suitable agent in the clinical management of inflamed cutaneous mycoses. Topical application of eberconazole was well tolerated in preclinical studies without any report of delayed hypersensitivity or photosensitivity reactions. There were no phototoxic effects. There was no significant systemic absorption. Animal toxicity studies have shown that it is safe, and the No Observed Effect Level was 2 ml/kg body weight in tested animals. It was not mutagenic and shared similar cytotoxicity profile with other imidazole antifungal products studied. Penetration studies using synthetic membranes revealed that eberconazole intrasets showed less variation as compared to clotrimazole and terbinafine intrasets. Overall amount of eberconazole released was more compared to comparators. In vitro and preclinical studies have demonstrated better therapeutic efficacy with eberconazole than clotrimazole and ketoconazole.

Antifungal mechanism of essential oil from Anethum graveolens seeds against Candida albicans

This work studied the antifungal mechanism of dill seed essential oil (DSEO) against Candida albicans. Flow cytometric analysis and inhibition of ergosterol synthesis were performed to clarify the mechanism of action of DSEO on C. albicans. Upon treatment of cells with DSEO, propidium iodide penetrated C. albicans through a lesion in its plasma membrane. DSEO also significantly reduced the amount of ergosterol. These findings indicate that the plasma membrane of C. albicans was damaged by DSEO. The effect of DSEO on the functions of the mitochondria in C. albicans was also studied. We assayed the mitochondrial membrane potential (mtΔψ) using rhodamine 123 and determined the production of mitochondrial dysfunction-induced reactive oxygen species (ROS) via flow cytometry. The effects of the antioxidant l-cysteine (Cys) on DSEO-induced ROS production and the antifungal effect of DSEO on C. albicans were investigated. Exposure to DSEO increased mtΔψ. Dysfunctions in the mitochondria caused ROS accumulation in C. albicans. This increase in the level of ROS production and DSEO-induced decrease in cell viability were prevented by the addition of Cys, indicating that ROS are an important mediator of the antifungal action of DSEO. These findings indicate that the cytoplasmic membrane and mitochondria are the main anti-Candida targets of DSEO.

Real-Time Microscopic Observation of Candida Biofilm Development and Effects Due to Micafungin and Fluconazole

To understand the process of Candida biofilm development and the effects of antifungal agents on biofilms, we analyzed real-time data comprising time-lapse images taken at times separated by brief intervals. The growth rate was calculated by measuring the change of biofilm thickness every hour. For the antifungal study, 5-h-old biofilms of Candida albicans were treated with either micafungin (MCFG) or fluconazole (FLCZ). MCFG began to suppress biofilm growth a few minutes after the initiation of the treatment, and this effect was maintained over the course of the observation period. In contrast, the suppressive effects of FLCZ on biofilm growth took longer to manifest: biofilms grew in the first 5 h after treatment, and then their growth was suppressed over the next 10 h, finally producing results similar to those observed with MCFG. MCFG was also involved in the disruption of cells in the biofilms, releasing string-like structures (undefined extracellular component) from the burst hyphae. Thus, MCFG inhibited the detachment of yeast cell clusters from the tips of hyphae. In contrast, FLCZ did not disrupt biofilm cells. MCFG also showed fast antifungal activity against Candida parapsilosis biofilms. In conclusion, our results show that inhibition of glucan synthesis due to MCFG contributed not only to fungicidal activity but also to the immediate suppression of biofilm growth, while FLCZ suppressed growth by inhibiting ergosterol synthesis. Therefore, those characteristic differences should be considered when treating clinical biofilm infections.

The mechanism of antifungal action of essential oil from dill (Anethum graveolens L.) on Aspergillus flavus.

The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus.

Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

BackgroundThe increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers.ResultsBased on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion.ConclusionsThe current study presents a systems biotechnology-based strategy for the engineering of the industrially important yeast P. pastoris combining the use of host specific DNA microarray technologies and physiological studies under well defined environmental conditions. Such studies allowed for the identification of novel targets related with protein trafficking and ergosterol biosynthesis for improved recombinant protein production. Nevertheless, further studies will be required to elucidate the precise mechanisms whereby membrane biogenesis and composition impact on protein secretion in P. pastoris.

The control of Aspergillus flavus with Cinnamomum jensenianum Hand.-Mazz essential oil and its potential use as a food preservative

The essential oil extracted from the bark of Cinnamomum jensenianum Hand.-Mazz was tested for antifungal activity against Aspergillus flavus. Fifty-five components accounting for 96.66% of the total oil composition were identified by GC–MS. The major components were eucalyptol (17.26%) and α-terpineol (12.52%). Mycelial growth and spore germination was inhibited by the oil in a dose-dependent manner. The oil also exhibited a noticeable inhibition on the dry mycelium weight and the synthesis of aflatoxin B1 (AFB1) by A. flavus, completely restraining AFB1 production at 6μl/ml. The possible mode of action of the oil against A. flavus is discussed based on changes in the mycelial ultrastructure. To confirm the target of the oil in the plasma membrane, studies on the inhibition of ergosterol synthesis were performed. Results show that the oil caused a considerable reduction in the ergosterol quantity. Thus, the essential oil from C. jensenianum can be used as a potential source for food preservative.

PdCYP51B, a new putative sterol 14α-demethylase gene of Penicillium digitatum involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis

Penicillium digitatum, causing green mold decay, is the most destructive postharvest pathogen of citrus fruits worldwide. The phenotypes and genotypes of 403 isolates of P. digitatum, collected from packing houses and supermarkets in Zhejiang, China, during 2000 to 2010, were characterized in terms of their imazalil sensitivity. The frequency of detected imazalil-resistant (IMZ-R) isolates increased from 2.1% in 2000 to 60-84% during 2005-2010. Only 6.5% and 4.5% of the collected IMZ-R isolates belong to the previously described IMZ-R1 and IMZ-R2 genotypes, respectively. To determine the resistance mechanism of the predominant and novel IMZ-R isolates of P. digitatum (termed IMZ-R3), genes PdCYP51B and PdCYP51C, homologous to the sterol 14α-demethylase encoded gene PdCYP51, were cloned from six IMZ-R3 and eight imazalil-sensitive (IMZ-S) isolates of P. digitatum. A unique 199-bp insertion was observed in the promoter region of PdCYP51B in all IMZ-R3 isolates examined but in none of the tested IMZ-S isolates. Further analysis by PCR confirmed that this insertion was present in all IMZ-R3 isolates but absent in IMZ-S, IMZ-R1, and IMZ-R2 isolates. Transcription levels of PdCYP51B in three IMZ-R3 isolates were found to be 7.5- to 13.6-fold higher than that in two IMZ-S isolates of P. digitatum. Introduction of another copy of PdCYP51B ( s ) (from IMZ-S) into an IMZ-S isolate decreased the sensitivity of P. digitatum to 14α-demethylation inhibitors (DMIs) only to a small extent, but introduction of a copy of PdCYP51B ( R ) (from IMZ-R3) dramatically increased the resistance level of P. digitatum to DMIs. Regarding PdCYP51C, no consistent changes in either nucleotide sequence or expression level were correlated with imazalil resistance among IMZ-R and IMZ-S isolates. Based on these results, we concluded that (1) the CYP51 family of P. digitatum contains the PdCYP51B and PdCYP51C genes, in addition to the known gene PdCYP51A (previously PdCYP51); (2) PdCYP51B is involved in DMI fungicide resistance; and (3) overexpression of PdCYP51B resulting from a 199-bp insertion mutation in the promoter region of PdCYP51B is responsible for the IMZ-R3 type of DMI resistance in P. digitatum.

Ketoconazole and miconazole alter potassium homeostasis in Saccharomyces cerevisiae

The effects of ketoconazole and miconazole uptake on K+ transport and the internal pH of Saccharomyces cerevisiae were studied. The uptake of both drugs was very fast, linear with concentration and not dependent on glucose, indicating entrance by diffusion and concentrating inside. Low (5.0μM) to intermediate concentrations (40μM) of both drugs produced a glucose-dependent K+ efflux; higher ones also produced a small influx of protons, probably through a K+/H+ exchanger, resulting in a decrease of the internal pH of the cells and the efflux of material absorbing at 260nm and phosphate. The cell membrane was not permeabilized. The K+ efflux with miconazole was dependent directly on the medium pH. This efflux results in an increased membrane potential, responsible for an increased Ca2+ uptake and other effects. These effects were not observed with two triazolic antifungals. A decrease of the Zeta (ζ) potential was observed at low concentrations of miconazole. Although the main effect of these antifungals is the inhibition of ergosterol synthesis, K+ efflux is an important additional effect to be considered in their therapeutic use. Under certain conditions, the use of single mutants of several transporters involved in the movements of K+ allowed to identify the participation of several antiporters in the efflux of the cation.

Inhibition of ergosterol synthesis by novel antifungal compounds targeting C-14 reductase

The limited number of clinically available antifungal drugs for life-threatening fungal infections has produced an increased demand for new agents. In the course of our screening for novel antifungals, we identified aminopiperidine derivatives which exhibit antifungal activities against the major pathogenic yeasts. Thin layer chromatography (TLC) analysis of the extracted non-saponifiable lipids from Candida albicans showed that these compounds inhibited the ergosterol production in the late step of the synthesis pathway. The results of an LC/Q-Tof MS analysis showed that abnormal sterols including predicted ignosterol, which is known to be accumulated in C. albicans ERG24 deleted mutant, were accumulated in C. albicans treated with one of these derivatives (Compound 1b). Furthermore, the partial disruption of the cell membrane of C. albicans treated with compound 1b was observed by electron microscopy analysis, suggesting its inhibition of ergosterol synthesis. Additionally, a genetic approach demonstrated that ERG24 gene would be responsible for the resistance of Saccharomyces cerevisiae against Compound 1b, strongly indicating that the enzyme targeted by Compound 1b is Erg24p. From all these data, we concluded that these aminopiperidine derivatives are novel antifungal compounds inhibiting C-14 reduction in the ergosterol synthesis pathway.