Direct Antifungal Effect Research Articles

Blap-6, a Novel Antifungal Peptide from the Chinese Medicinal Beetle Blaps rhynchopetera against Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans) is a pathogenic fungus that can cause life-threatening meningitis, particularly in individuals with compromised immune systems. The current standard treatment involves the combination of amphotericin B and azole drugs, but this regimen often leads to inevitable toxicity in patients. Therefore, there is an urgent need to develop new antifungal drugs with improved safety profiles. We screened antimicrobial peptides from the hemolymph transcriptome of Blaps rhynchopetera (B. rhynchopetera), a folk Chinese medicine. We found an antimicrobial peptide named blap-6 that exhibited potent activity against bacteria and fungi. Blap-6 is composed of 17 amino acids (KRCRFRIYRWGFPRRRF), and it has excellent antifungal activity against C. neoformans, with a minimum inhibitory concentration (MIC) of 0.81 μM. Blap-6 exhibits strong antifungal kinetic characteristics. Mechanistic studies revealed that blap-6 exerts its antifungal activity by penetrating and disrupting the integrity of the fungal cell membrane. In addition to its direct antifungal effect, blap-6 showed strong biofilm inhibition and scavenging activity. Notably, the peptide exhibited low hemolytic and cytotoxicity to human cells and may be a potential candidate antimicrobial drug for fungal infection caused by C. neoformans.

Discovery of Pyrazolone Carbothioamide Derivatives as Inhibitors of the Pdr1-KIX Interaction for Combinational Treatment of Azole-Resistant Candidiasis.

Candida glabrata has emerged as an important opportunistic pathogen of invasive candidiasis due to increasing drug resistance. Targeting Pdr1-KIX interactions with small molecules represents a potential strategy for treating drug-resistant candidiasis. However, effective Pdr1-KIX inhibitors are rather limited, hindering the validation of target druggability. Here, new Pdr1-KIX inhibitors were designed and assayed. Particularly, compound B8 possessed a new chemical scaffold and exhibited potent KIX binding affinity, leading to enhanced synergistic efficacy with fluconazole to treat resistant C. glabrata infection (FICI = 0.28). Compound B8 acted by inhibiting the efflux pump and down-regulating resistance-associated genes through blocking the Pdr1-KIX interaction. Compound B8 exhibited excellent in vitro and in vivo antifungal potency in combination with fluconazole against azole-resistant C. glabrata. It also had direct antifungal effect to treat C. glabrata infection, suggesting new mechanisms of action independent of Pdr1-KIX inhibition. Therefore, compound B8 represents a promising lead compound for antifungal drug development.

Endophytic Bacillus atrophaeus CHGP13 and salicylic acid inhibit blue mold of lemon by regulating defense enzymes.

Lemons (Citrus limon L.) are one of the most economically important and consumed fruit worldwide. The species is vulnerable to several postharvest decay pathogens, of which Penicillium italicum associated with blue mold disease is the most damaging. This study investigates the use of integrated management for blue mold of lemon using lipopeptides (LPs) extracted from endophytic Bacillus strains and resistance inducers. Two resistance inducers; salicylic acid (SA) and benzoic acid (BA) were tested at 2, 3, 4, and 5 mM concentrations against the development of blue mold on lemon fruit. The 5 mM SA treatment produced the lowest disease incidence (60%) and lesion diameter (1.4 cm) of blue mold on lemon fruit relative to the control. In an in vitro antagonism assay eighteen Bacillus strains were evaluated for their direct antifungal effect against P. italicum; CHGP13 and CHGP17 had the greatest inhibition zones of 2.30 and 2.14 cm. Lipopeptides (LPs) extracted from CHGP13 and CHGP17 also inhibited the colony growth of P. italicum. LPs extracted from CHGP13 and 5 mM SA were tested as single and combined treatments against disease incidence and lesion diameter of blue mold on lemon fruit. SA + CHGP13 + PI had the lowest disease incidence (30%) and lesion diameter (0.4 cm) of P. italicum on lemon fruit relative to the other treatments. Furthermore, the lemon fruit treated with SA + CHGP13 + PI had the highest PPO, POD, and PAL activities. The postharvest quality analysis of the lemon fruit including fruit firmness, total soluble solids, weight loss, titratable acidity, and ascorbic acid content revealed that the treatment SA + CHGP13 + PI had little effect on fruit quality compared to the healthy control. These findings indicate that Bacillus strains and resistance inducers can be used as components of integrated disease management for the blue mold of lemon.

Vitamin D3: A promising antifungal and antibiofilm agent against Candida species.

Candida species are opportunistic fungal pathogens that cause mild to life-threatening infections in both immunocompetent and immunocompromised populations. The increasing prevalence of drug-resistant Candida species has posed a significant challenge to the management of infections in clinical settings. Therefore, this study aimed to investigate the direct antifungal and antibiofilm effect of vitamin D3 against Candida species. The antifungal activity of vitamin D3 was evaluated by broth microdilution method based on the Clinical and Laboratory Standard Institute. Prevention of biofilm formation by Candida albicans was measured using the XTT assay following exposure to different concentrations of vitamin D3. Moreover, expression of Agglutinin-like sequence gene 1 (ALS1), hyphal wall protein gene (HWP1), secreted aspartyl proteinase 6 gene (SAP6), and morphogenesis pathway regulatory gene (EFG1) were analyzed by real-time polymerase chain reaction using the comparative Ct method (ΔΔ Ct) after exposure to vitamin D3. Vitamin D3 showed antifungal activity against Candida species ranging from 1-128 μg/mL. Furthermore, vitamin D3 inhibited biofilm formation in a dose-dependent manner, with IC50 of 7.5 μg/mL. Treatment with vitamin D3 resulted in significant upregulation of the EFG1, ALS1, and SAP6 genes under hypha-inducing conditions to overcome environmental challenges. Results of the current study demonstrated that vitamin D3 has a significant inhibitory effect on Candida growth and biofilm formation. Considering its demonstrated antifungal and antibiofilm properties, vitamin D3 holds promise as a potential agent for medical applications.

Biochar for the Control of Plant Pathogens

Biochar, the solid product of pyrolysis of biomass at thermal degradation temperatures, is useful in agriculture as manure for enhancing plant growth through the supply of nutrients. It is used in protected cultivation practices of vegetables and flower crops in the pot culture and grow bags especially to improve soil physicochemical properties, and in hydroponics to remove pollutants like heavy metals in the water. The usage of biochar as a potential soil amendment for plant growth promotion, improving soil fertility and plant disease suppression are being explored in recent years. Biochar made from many of the agro waste materials was found to suppress the plant pathogens in the soil and also effective in controlling the pathogens affecting aerial parts of the plants. Although direct antifungal or antibacterial effects and metabolites of biochar are poorly understood, induced systemic resistance in plants through signal transduction and expression of defence chemicals and metabolites have been studied. In addition, microbiome analyses through metagenome sequencing revealed an increase in the population of beneficial microbes (antagonistic to plant pathogens) in the rhizosphere soils applied with biochar.

Visible-Light-Activated Molecular Machines Kill Fungi by Necrosis Following Mitochondrial Dysfunction and Calcium Overload.

Invasive fungal infections are a growing public health threat. As fungi become increasingly resistant to existing drugs, new antifungals are urgently needed. Here, it is reported that 405-nm-visible-light-activated synthetic molecular machines (MMs) eliminate planktonic and biofilm fungal populations more effectively than conventional antifungals without resistance development. Mechanism-of-action studies show that MMs bind to fungal mitochondrial phospholipids. Upon visible light activation, rapid unidirectional drilling of MMs at ≈3 million cycles per second (MHz) results in mitochondrial dysfunction, calcium overload, and ultimately necrosis. Besides their direct antifungal effect, MMs synergize with conventional antifungals by impairing the activity of energy-dependent efflux pumps. Finally, MMs potentiate standard antifungals both in vivo and in an ex vivo porcine model of onychomycosis, reducing the fungal burden associated with infection.

CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models.

Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus-specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.

Fructan oligosaccharide priming alters apoplastic sugar dynamics and improves resistance against Botrytis cinerea in chicory.

Carbohydrates such as fructans can be involved in priming or defence stimulation, and hence potentially provide new strategies for crop protection against biotic stress. Chicory (Cichorium intybus) is a model plant for fructan research and is a crop with many known health benefits. Using the chicory-Botrytis cinerea pathosystem, we tested the effectiveness of fructan-induced immunity, focussing on different plant and microbial fructans. Sugar dynamics were followed after priming and subsequent pathogen infection. Our results indicated that many higher plants might detect extracellular levan oligosaccharides (LOS) of microbial origin, while chicory also detects extracellular small inulin-type fructooligosaccharides (FOS) of endogenous origin, thus differing from the findings of previous fructan priming studies. No clear positive effects were observed for inulin or mixed-type fructans. An elicitor-specific burst of reactive oxygen species was observed for sulfated LOS, while FOS and LOS both behaved as genuine priming agents. In addition, a direct antifungal effect was observed for sulfated LOS. Intriguingly, LOS priming led to a temporary increase in apoplastic sugar concentrations, mainly glucose, which could trigger downstream responses. Total sugar and starch contents in total extracts of LOS-primed leaves were higher after leaf detachment, indicating they could maintain their metabolic activity. Our results indicate the importance of balancing intra- and extracellular sugar levels (osmotic balance) in the context of 'sweet immunity' pathways.

Antifungals influence the immune-related transcriptomic landscape of human monocytes after Aspergillus fumigatus infection

The heterogeneity of clinical responses to antifungals in aspergillosis is partially understood. We hypothesized that besides direct antifungal effects, these discrepancies may be related to different immunomodulatory profiles. Human THP-1 monocytes were coincubated in vitro with Aspergillus fumigatus and variable concentrations of voriconazole (0.5, 1 and 2 mg/l), caspofungin (1 and 2 mg/l), amphotericin B deoxycholate (0.25, 0.5 and 1 mg/l) and liposomal amphotericin B (1, 2 and 3 mg/l). After 6 h of coincubation, total cellular RNA was extracted, converted into cDNA, and transcription of 84 genes involved in antifungal immunity was measured through RT-qPCR. The presence of A. fumigatus was the main driver of the global immune-related transcriptomic response. After Aspergillus infection, thirty genes were upregulated, while 19 genes were downregulated. Discrepancies across antifungals were also evident; voriconazole-containing conditions showed similar reaction to natural infection, while the use of liposomal Amphotericin B significantly decreased the inflammatory response. Chemokines (notably CCL20 and CXCL2) and pro-inflammatory cytokines (IL1A, IL1B, IL23, G-CSF) exhibited the most pronounced differences across antifungals. Pattern recognition receptors and adaptor protein transcription were minimally affected. Protein–protein-interaction network analysis showed that IL23A played a dominant role in upregulated genes. Pathway enrichment analysis indicated that cytokine-cytokine receptor integration, TNF signaling pathways and Toll-like receptor pathways were highly involved. This exploratory study confirms the heterogeneous immunomodulatory role of antifungals. Overall, voriconazole appears to maintain an early pro-inflammatory response seen in natural infection. Assessment of immunomodulatory response with clinical response may provide a better rationale for differences observed across antifungals.

Brazilian Red Propolis shows antifungal and immunomodulatory activities against Paracoccidioides brasiliensis

Ethnopharmacological relevanceParacoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in South America and especially in Brazil with severe clinical consequences that need broadened therapeutic options.Propolis is a natural resin from bees used in folk medicine for centuries with the first report in the ancient history of Egypt by Eberly papyrus, in Middle-Ages used to wash the newborn's umbilical cord and World War II as antiseptic or antibiotics. Nowadays it is a natural product worldwide consumed as food and traditionally used for oral and systemic diseases as an anti-inflammatory, antimicrobial, antifungal, and other diseases. Brazilian red propolis (BRP) is a new type of propolis with a distinguished chemical profile and biological activities from propolis (green) with pharmacological properties such as antimicrobial, anti-inflammatory, antioxidant, and others. Aim of studyThus, the main purpose of this study was to investigate the direct in vitro and ex vivo effect of BRP on Paracoccidioides brasiliensis. Material and methodsAntifungal activity of different concentrations of BRP on a virulent P. brasiliensis isolate (Pb18) was evaluated using the microdilution technique. Also, mice splenic cells co-cultured with Pb18 were treated with BRP at different times and concentrations (only Pb18 = negative control). Mice were inoculated with Pb18 and treated with different concentrations of BRP (50–500 mg/mL) in a subcutaneous air pouch. In this later experimental model, macroscopic characteristics of the air pouch were evaluated, and cellular exudate was collected and analyzed for cellular composition, mitochondrial activity, total protein reactive oxygen specimens (ROS), and nitric oxide production, as well as the number of viable fungal cells. ResultsThe in vitro experiments showed remarkable direct antifungal activity of BRP, mainly with the highest concentration employed (500 mg/mL), reducing the number of viable cells to 10% of the original inoculum after 72 h incubation. The splenocytes co-cultivation assays showed that BRP had no cytotoxic effect on these cells, on the contrary, exerted a stimulatory effect. This stimulation was also observed on the PMNs at the air pouch, as verified by production of ROS and total proteins and mitochondrial activity. This activation resulted in enhanced fungicidal activity, mainly with the 500 mg/mL concentration of BRP. An anti-inflammatory effect was also detected, as verified by the smaller volume of the BRP-treated air pouch as well as by an earlier shift from neutrophils to mononuclear cells present in the infection site. ConclusionOur results strongly suggest, for the first time in the literature, that Brazilian Red propolis has four protective mechanisms in experimental paracoccidioidomycosis: activating neutrophils, exerting a direct antifungal effect, preventing fungal dissemination, and controlling excessive inflammation process.

Saccharin Provides Protection and Activates Defense Mechanisms in Wheat Against the Hemibiotrophic Pathogen Zymoseptoria tritici.

Plant resistance inducers are among the most promising alternatives to develop sustainable crop protection. Here, we examined the ability of saccharin, a metabolite derived from probenazole, to protect wheat against Zymoseptoria tritici, the most frequently occurring and damaging foliar pathogen on this crop. The experiments were performed in the greenhouse by treating seedlings of the wheat cultivar 'Alixan' with 15 mM of saccharin 2 days before challenge inoculation with the Z. tritici pathogenic strain T02596. Foliar application of saccharin resulted in 77% lower disease severity than in nontreated control plants. In vitro and in planta assays showed that saccharin did not exhibit any direct antifungal effect on spore germination or hyphal growth. Molecular investigations from 2 to 7 days posttreatment (dpt) revealed that saccharin treatment upregulates the expression of genes encoding for lipoxygenase (LOX) at all sampled time points and pathogenesis-related protein 1 (PR1) at 7 dpt, in both noninfectious and infectious contexts, as well as peroxidase (POX2) in noninfectious conditions. However, saccharin did not induce significant change in the expression of PAL gene encoding for phenylalanine ammonia-lyase. Our findings report for the first time the potential of saccharin to confer protection in wheat against Z. tritici through an elicitation and priming of LOX and PR gene-related defense pathways. Additional investigations would provide a better deciphering of defense mechanisms activated by this molecule in wheat against Z. tritici.

Synthetic Mono-Rhamnolipids Display Direct Antifungal Effects and Trigger an Innate Immune Response in Tomato against Botrytis Cinerea.

Natural rhamnolipids are potential biocontrol agents for plant protection against bacterial and fungal diseases. In this work, we synthetized new synthetic mono-rhamnolipids (smRLs) consisting in a rhamnose connected to a simple acyl chain and differing by the nature of the link and the length of the lipid tail. We then investigated the effects of these ether, ester, carbamate or succinate smRL derivatives on Botrytis cinerea development, symptoms spreading on tomato leaves and immune responses in tomato plants. Our results demonstrate that synthetic smRLs are able to trigger early and late immunity-related plant defense responses in tomato and increase plant resistance against B. cinerea in controlled conditions. Structure-function analysis showed that chain length of the lipidic part and type of acyl chain were critical to smRLs immune activity and to the extent of symptoms caused by the fungus on tomato leaves.

Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian β-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.

Transcriptomic Analysis of Avocado Hass (Persea americana Mill) in the Interaction System Fruit-Chitosan-Colletotrichum.

Avocado (Persea americana) is one of the most important crops in Mexico as it is the main producer, consumer, and exporter of avocado fruit in the world. However, successful avocado commercialization is often reduced by large postharvest losses due to Colletotrichum sp., the causal agent of anthracnose. Chitosan is known to have a direct antifungal effect and acts also as an elicitor capable of stimulating a defense response in plants. However, there is little information regarding the genes that are either activated or repressed in fruits treated with chitosan. The aim of this study was to identify by RNA-seq the genes differentially regulated by the action of low molecular weight chitosan in the avocado-chitosan-Colletotrichum interaction system. The samples for RNA-seq were obtained from fruits treated with chitosan, fruits inoculated with Colletotrichum and fruits both treated with chitosan and inoculated with the fungus. Non-treated and non-inoculated fruits were also analyzed. Expression profiles showed that in short times, the fruit-chitosan system presented a greater number of differentially expressed genes, compared to the fruit-pathogen system. Gene Ontology analysis of differentially expressed genes showed a large number of metabolic processes regulated by chitosan, including those preventing the spread of Colletotrichum. It was also found that there is a high correlation between the expression of genes in silico and qPCR of several genes involved in different metabolic pathways.

Stryphnodendron adstringens and purified tannin on Pythium insidiosum: in vitro and in vivo studies

BackgroundPythium insidiosum is the etiological agent of pythiosis, an emerging life-threatening infectious disease in tropical and subtropical regions. The pathogen is a fungus-like organism resistant to antifungal therapy, for this reason, most cases need extensive surgical debridments as treatment, but depending on the size and anatomical region of the lesion, such approach is unfeasible. We investigate the fungicidal effect and toxicity of crude bark extract of Stryphnodendron adstringens and commercially available tannin on Pythium insidiosum both in vitro and in vivo.MethodsStandardized fragments of mycelia of fifteen isolates of P. insidiosum were tested with different concentrations of bark extract (10 to 30% v/v) and tannin (0.5, 1.0 and 1.5 mg/mL). For in vivo study, fifteen rabbits were experimentally infected with zoospores of P. insidiosum and treated by oral and intralesional applications of bark extract and tannin. Acute toxicity tests with both substances were also performed in rats.ResultsIn vitro studies showed fungicidal effect for both substances at different concentrations and the SEM showed alteration on the cell wall surface of the pathogen. All infected rabbits developed a firm nodular mass that reached around 90 mm2 ninety days after inoculation, but neither the intralesional inoculation of tannin, nor the oral administration of crude extract and tannin were able to promote remission of the lesions.ConclusionsLesions developed by rabbits presented an encapsulated abscess being quite different of naturally acquired pythiosis, which is characterized by ulcerated lesions. Since no toxicity was observed in rats or rabbits inoculated with these products, while in vitro experiments showed direct antifungal effect, therapeutic activity of S. adstringens and tannin should be clinically tested as an alternative for healing wounds in naturally acquired pythiosis.

Ketoconazole inhibits Malassezia furfur morphogenesis in vitro under filamentation optimized conditions.

Malassezia furfur, a constituent of the normal human skin flora, is an etiological agent of pityriasis versicolor, which represents one of the most common human skin diseases. Under certain conditions, both exogenous and endogenous, the fungus can transition from a yeast form to a pathogenic mycelial form. To develop a standardized medium for reproducible production of the mycelial form of M. furfur to develop and optimize susceptibility testing for this pathogen, we examined and characterized variables, including kojic acid and glycine concentration, agar percentage, and pH, to generate a chemically defined minimal medium on which specific inoculums of M. furfur generated the most robust filamentation. Next, we examined the capacity of ketoconazole to inhibit the formation of M. furfur mycelial form. Both low and high, 0.01, 0.05 and 0.1µg/ml concentrations of ketoconazole significantly inhibited filamentation at 11.9, 54.5 and 86.7%, respectively. Although ketoconazole can have a direct antifungal effect on both M. furfur yeast and mycelial cells, ketoconazole also has a dramatic impact on suppressing morphogenesis. Since mycelia typified the pathogenic form of Malassezia infection, the capacity of ketoconazole to block morphogenesis may represent an additional important effect of the antifungal.

Burkholderia phytofirmans PsJN Confers Grapevine Resistance against Botrytis cinerea via a Direct Antimicrobial Effect Combined with a Better Resource Mobilization.

Plant innate immunity serves as a surveillance system by providing the first line of powerful weapons to fight against pathogen attacks. Beneficial microorganisms and Microbial-Associated Molecular Patterns might act as signals to trigger this immunity. Burkholderia phytofirmans PsJN, a highly efficient plant beneficial endophytic bacterium, promotes growth in a wide variety of plants including grapevine. Further, the bacterium induces plant resistance against abiotic and biotic stresses. However, no study has deciphered triggered-mechanisms during the tripartite interaction between grapevine, B. phytofirmans PsJN and Botrytis cinerea. Herein, we showed that in contrast with classical rhizobacteria, which are restricted in the root system and act through ISR, B. phytofirmans PsJN is able to migrate until aerial part and forms at leaves surface a biofilm around B. cinerea mycelium to restrict the pathogen. Nevertheless, considering the endophytic level of PsJN in leaves, the plant protection efficacy of B. phytofirmans PsJN could not be explained solely by its direct antifungal effect. Deeper investigations showed a callose deposition, H2O2 production and primed expression of PR1, PR2, PR5, and JAZ only in bacterized-plantlets after pathogen challenge. The presence of PsJN modulated changes in leaf carbohydrate metabolism including gene expression, sugar levels, and chlorophyll fluorescence imaging after Botrytis challenge. Our findings indicated that protection induced by B. phytofirmans PsJN was multifaceted and relied on a direct antifungal effect, priming of defense mechanisms as well as the mobilization of carbon sources in grapevine leaf tissues.

ARACINs, Brassicaceae-specific peptides exhibiting antifungal activities against necrotrophic pathogens in Arabidopsis.

Plants have developed a variety of mechanisms to cope with abiotic and biotic stresses. In a previous subcellular localization study of hydrogen peroxide-responsive proteins, two peptides with an unknown function (designated ARACIN1 and ARACIN2) have been identified. These peptides are structurally very similar but are transcriptionally differentially regulated during abiotic stresses during Botrytis cinerea infection or after benzothiadiazole and methyl jasmonate treatments. In Arabidopsis (Arabidopsis thaliana), these paralogous genes are positioned in tandem within a cluster of pathogen defense-related genes. Both ARACINs are small, cationic, and hydrophobic peptides, known characteristics for antimicrobial peptides. Their genes are expressed in peripheral cell layers prone to pathogen entry and are lineage specific to the Brassicaceae family. In vitro bioassays demonstrated that both ARACIN peptides have a direct antifungal effect against the agronomically and economically important necrotrophic fungi B. cinerea, Alternaria brassicicola, Fusarium graminearum, and Sclerotinia sclerotiorum and yeast (Saccharomyces cerevisiae). In addition, transgenic Arabidopsis plants that ectopically express ARACIN1 are protected better against infections with both B. cinerea and A. brassicicola. Therefore, we can conclude that both ARACINs act as antimicrobial peptides.

Sodium carbonate and bicarbonate treatments induce resistance to postharvest green mould on citrus fruit

The aim of this study was to investigate the ability of two salts, sodium carbonate and bicarbonate, to activate defence mechanisms in citrus fruit against postharvest green mould caused by Penicillium digitatum. In particular, once there was confirmed salt antifungal activity in the absence of direct contact with the pathogen, changes in enzyme activity and expression levels of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase (PAL), and phytoalexin (scoparone, scopoletin, umbelliferone) and sugar (glucose, fructose, sucrose) contents in treated oranges were analyzed. Overall, sodium carbonate and bicarbonate increases the activity of β-1,3-glucanase, peroxidase, and PAL enzymes in orange tissues. Gene expression analyses confirmed PAL up-regulation particularly 12h after treatment application. HPLC analyses of peel extracts showed increased amounts of the sugars and phytoalexins, compared to control tissues, with sucrose and scoparone being the most represented. The results suggest that, although salts exert a direct antifungal effect on P. digitatum, they are also able to induce citrus fruit defence mechanisms to postharvest decay. The defence response seems correlated with the up-regulation of the phenylpropanoid pathway, which has a role in the adaptation to various stresses. This response could result in natural reaction to wounding and pathogen attack in citrus, enhancing its protective effect. As a consequence, the fruit might have a better chance of successful defence against the decay.

Chloroquine Modulates the Fungal Immune Response in Phagocytic Cells From Patients With Chronic Granulomatous Disease

Invasive aspergillosis is a major threat to patients with chronic granulomatous disease (CGD). Fungal pathogenesis is the result of a diminished antifungal capacity and dysregulated inflammation. A deficient NADPH-oxidase complex results in defective phagolysosomal alkalization. To investigate the contribution of defective pH regulation in phagocytes among patients with CGD during fungal pathogenesis, we evaluated the effect of the acidotropic, antimalarial drug chloroquine (CQ) on the antifungal capacity of polymorphonuclear cells (PMNs) and on the inflammatory response of peripheral blood mononuclear cells (PBMCs). Chloroquine exerted a direct pH-dependent antifungal effect on Aspergillus fumigatus and Aspergillus nidulans; it increased the antifungal activity of PMNs from patients with CGD at a significantly lower concentration, compared with the concentration for PMNs from healthy individuals; and decreased the hyperinflammatory state of PBMCs from patients with CGD, as observed by decreased tumor necrosis factor α and interleukin 1β release. Chloroquine targets both limbs of fungal pathogenesis and might be of great value in the clearance of invasive aspergillosis in patients with CGD.