Antifungal activity of ß-lapachone against a fluconazole-resistant Candida auris strain.

Candida spp. can be found in the human microbiome. However, immunocompromised patients are likely to develop invasive Candida infections, with mortality rates higher than 50%. The discovery of C. auris, a species that rapidly acquire antifungal resistance, increased the concern about Candida infections. The limited number of antifungal agents and the high incidence of resistance to them make imperative the development of new antifungal drugs. ß-lapachone is a biological active naphthoquinone that displays antifungal activity against C. albicans and C. glabrata. The aim of this study was to evaluate if this substance affects C. auris growth and elucidate its mechanism of action. A fluconazole-resistant C. auris isolate was used in this study. The antifungal activity of ß-lapachone was determined through microbroth dilution assays, and its mechanism of action was evaluated using fluorescent probes. Interaction with fluconazole and amphotericin B was assessed by disk diffusion assay and checkerboard. ß-lapachone inhibited planktonic C. auris cell growth by 92.7%, biofilm formation by 84.9%, and decrease the metabolism of preformed biofilms by 87.1% at 100 µg/ml. At 100 µg/ml, reductions of 30% and 59% of Calcofluor White and Nile red fluorescences were observed, indicating that ß-lapachone affects cell wall chitin and neutral lipids content, respectively. Also, the ratio 590 nm/529 nm of JC-1 decreased 52%, showing that the compound affects mitochondria. No synergism was observed between ß-lapachone and fluconazole or amphotericin B. Data show that ß-lapachone may be a promising candidate to be used as monotherapy to treat C. auris resistant infections.

Miltefosine: A Repurposing Drug against Mucorales Pathogens.

Mucorales are a group of non-septated filamentous fungi widely distributed in nature, frequently associated with human infections, and are intrinsically resistant to many antifungal drugs. For these reasons, there is an urgent need to improve the clinical management of mucormycosis. Miltefosine, which is a phospholipid analogue of alkylphosphocholine, has been considered a promising repurposing drug to be used to treat fungal infections. In the present study, miltefosine displayed antifungal activity against a variety of Mucorales species, and it was also active against biofilms formed by these fungi. Treatment with miltefosine revealed modifications of cell wall components, neutral lipids, mitochondrial membrane potential, cell morphology, and the induction of oxidative stress. Treated Mucorales cells also presented an increased susceptibility to SDS. Purified ergosterol and glucosylceramide added to the culture medium increased miltefosine MIC, suggesting its interaction with fungal lipids. These data contribute to elucidating the effect of a promising drug repurposed to act against some relevant fungal pathogens that significantly impact public health.

Structural and Functional Alterations Caused by Aureobasidin A in Clinical Resistant Strains of Candida spp.

Candida species are one of the most concerning causative agents of fungal infections in humans. The treatment of invasive Candida infections is based on the use of fluconazole, but the emergence of resistant isolates has been an increasing concern which has led to the study of alternative drugs with antifungal activity. Sphingolipids have been considered a promising target due to their roles in fungal growth and virulence. Inhibitors of the sphingolipid biosynthetic pathway have been described to display antifungal properties, such as myriocin and aureobasidin A, which are active against resistant Candida isolates. In the present study, aureobasidin A did not display antibiofilm activity nor synergism with amphotericin B, but its combination with fluconazole was effective against Candida biofilms and protected the host in an in vivo infection model. Alterations in treated cells revealed increased oxidative stress, reduced mitochondrial membrane potential and chitin content, as well as altered morphology, enhanced DNA leakage and a greater susceptibility to sodium dodecyl sulphate (SDS). In addition, it seems to inhibit the efflux pump CaCdr2p. All these data contribute to elucidating the role of aureobasidin A on fungal cells, especially evidencing its promising use in clinical resistant isolates of Candida species.

An Alpha-Glucan from Lomentospora prolificans Mediates Fungal-Host Interaction Signaling through Dectin-1 and Mincle.

Scedosporium and Lomentospora are a group of filamentous fungi with some clinically relevant species causing either localized, invasive, or disseminated infections. Understanding how the host immune response is activated and how fungi interact with the host is crucial for a better management of the infection. In this context, an α-glucan has already been described in S. boydii, which plays a role in the inflammatory response. In the present study, an α-glucan has been characterized in L. prolificans and was shown to be exposed on the fungal surface. The α-glucan is recognized by peritoneal macrophages and induces oxidative burst in activated phagocytes. Its recognition by macrophages is mediated by receptors that include Dectin-1 and Mincle, but not TLR2 and TLR4. These results contribute to the understanding of how Scedosporium's and Lomentospora's physiopathologies are developed in patients suffering with scedosporiosis and lomentosporiosis.

Pandemic Response Box® library as a source of antifungal drugs against Scedosporium and Lomentospora species.

Scedosporium and Lomentospora species are opportunistic filamentous fungi that cause localized and disseminated infections in immunocompetent and immunocompromised patients. These species are considered resistant fungi due to their low susceptibility to most current antifungal agents used in healthcare settings. The search for new compounds that could work as promising candidate antifungal drugs is an increasing field of interest. In this context, in the present study we screened the Pandemic Response Box® library (Medicines for Malaria Venture [MMV], Switzerland) to identify compounds with antifungal activity against Scedosporium and Lomentospora species. An initial screening of the drugs from this collection at 5 µM was performed using a clinical Scedosporium aurantiacum isolate according to the EUCAST protocol. Compounds with activity against this fungus were also tested against four other species (S. boydii¸ S. dehoogii, S. apiospermum and L. prolificans) at concentrations ranging from 0.078 to 10 µM. Seven compounds inhibited more than 80% of S. aurantiacum growth, three of them (alexidine, amorolfine and olorofim) were selected due to their differences in mechanism of action, especially when compared to drugs from the azole class. These compounds were more active against biofilm formation than against preformed biofilm in Scedosporium and Lomentospora species, except alexidine, which was able to decrease preformed biofilm about 50%. Analysis of the potential synergism of these compounds with voriconazole and caspofungin was performed by the checkerboard method for S. aurantiacum. The analysis by Bliss methodology revealed synergistic effects among selected drugs with caspofungin. When these drugs were combined with voriconazole, only alexidine and amorolfine showed a synergistic effect, whereas olorofim showed an antagonistic effect. Scanning electron microscopy revealed that alexidine induces morphology alterations in S. aurantiacum biofilm grown on a catheter surface. Reactive oxygen species production, mitochondrial activity and surface components were analyzed by fluorescent probes when S. aurantiacum was treated with selected drugs and revealed that some cell parameters are altered by these compounds. In conclusion, alexidine, amorolfine and olorofim were identified as promising compounds to be studied against scedosporiosis and lomentosporiosis.

Promising Antifungal Molecules against Mucormycosis Agents Identified from Pandemic Response Box®: In Vitro and In Silico Analyses.

Mucormycosis is considered concerning invasive fungal infections due to its high mortality rates, difficult diagnosis and limited treatment approaches. Mucorales species are highly resistant to many antifungal agents and the search for alternatives is an urgent need. In the present study, a library with 400 compounds called the Pandemic Response Box® was used and four compounds were identified: alexidine and three non-commercial molecules. These compounds showed anti-biofilm activity, as well as alterations in fungal morphology and cell wall and plasma membrane structure. They also induced oxidative stress and mitochondrial membrane depolarization. In silico analysis revealed promising pharmacological parameters. These results suggest that these four compounds are potent candidates to be considered in future studies for the development of new approaches to treat mucormycosis.

Professor Luiz R. Travassos and the study of surface structures of fungal pathogens.

Brazilian medical mycology considerably expanded in the last decades due to the efforts of several pioneers who started and expanded mycology during the twentieth century. In this manuscript, we highlight some of the contributions of one of these pioneers: Professor Luiz R. Travassos, who started his career in the field of microbiology in the 1960s. We will discuss his contributions to the areas of medical mycology and glycobiology, with a focus on glycosphingolipids, sialic acids, and surface enzymes.

Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic Sporothrix Species.

The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis, S. schenckii, and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.

Digoxin Derivatives Sensitize a Saccharomyces cerevisiae Mutant Strain to Fluconazole by Inhibiting Pdr5p.

The poor outcome of treatments for fungal infections is a consequence of the increasing incidence of resistance to antifungal agents, mainly due to the overexpression of efflux pumps. To surpass this mechanism of resistance, a substance able to inhibit these pumps could be administered in association with antifungals. Saccharomyces cerevisiae possesses an efflux pump (Pdr5p) homologue to those found in pathogenic yeast. Digoxin is a natural product that inhibits Na+, K+-ATPase. The aim of this study was to evaluate whether digoxin and its derivatives (i.e., DGB, digoxin benzylidene) can inhibit Pdr5p, reversing the resistance to fluconazole in yeasts. An S. cerevisiae mutant strain that overexpresses Pdr5p was used in the assays. The effects of the compounds on yeast growth, efflux activity, and Pdr5p ATPase activity were measured. All derivatives enhanced the antifungal activity of fluconazole against S. cerevisiae, in comparison to fluconazole alone, with FICI values ranging from 0.031 to 0.500. DGB 1 and DGB 3 presented combined effects with fluconazole against a Candida albicans strain, with fractional inhibitory concentration index (FICI) values of 0.625 and 0.281, respectively The compounds also inhibited the efflux of rhodamine 6G and Pdr5p ATPase activity, with IC50 values ranging from 0.41 µM to 3.72 µM. The results suggest that digoxin derivatives impair Pdr5p activity. Considering the homology between Pdr5p and efflux pumps from pathogenic fungi, these compounds are potential candidates to be used in association with fluconazole to treat resistant fungal infections.

Characterization of Aspergillus nidulans Biofilm Formation and Structure and Their Inhibition by Pea Defensin Psd2.

Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp. The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Psd2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Psd2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Psd2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 µM Psd2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Psd2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Psd2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.

Identification of Promising Antifungal Drugs against Scedosporium and Lomentospora Species after Screening of Pathogen Box Library.

Fungal infections have been increasing during the last decades. Scedosporium and Lomentospora species are filamentous fungi most associated to those infections, especially in immunocompromised patients. Considering the limited options of treatment and the emergence of resistant isolates, an increasing concern motivates the development of new therapeutic alternatives. In this context, the present study screened the Pathogen Box library to identify compounds with antifungal activity against Scedosporium and Lomentospora. Using antifungal susceptibility tests, biofilm analysis, scanning electron microscopy (SEM), and synergism assay, auranofin and iodoquinol were found to present promising repurposing applications. Both compounds were active against different Scedosporium and Lomentospora, including planktonic cells and biofilm. SEM revealed morphological alterations and synergism analysis showed that both drugs present positive interactions with voriconazole, fluconazole, and caspofungin. These data suggest that auranofin and iodoquinol are promising compounds to be studied as repurposing approaches against scedosporiosis and lomentosporiosis.

Development and Immunogenicity of a Brazilian Glycoconjugate vaccine against Meningococcal W in a Pilot Scale.

Recent changes in the epidemiology of meningococcal have been reported and meningococcal group W (MenW) has become the third most prevalent group isolated in Brazil in the last 10 years. In this study we have developed a conjugate vaccine for MenW using a modified reductive amination conjugation method through a covalent linkage between periodate-oxidized MenW non-O-acetylated polysaccharide and hydrazide-activated monomeric tetanus toxoid. Process control of bulks was done by physicochemical analysis including polysaccharide and protein quantification, high performance liquid chromatography - size exclusion chromatography, capillary electrophoresis, and hydrogen nuclear magnetic resonance. Conjugate bulks were best produced with concentration of polysaccharide twice as high as protein, at room temperature, and pH approximately 6.0. A scaled-up bulk (100 mg scale) was formulated and inoculated intramuscularly in mice in a dose-response study (0.1, 0.5, 1.0 and 10.0 µg of polysaccharide/dose). The immunogenicity of conjugate bulks was determined by serum bactericidal assay and ELISA assays of serum from immunized mice. ELISA and SBA titers revealed high titers of IgG and demonstrated the functionality of the antibodies produced in all doses studied 15 days after the third dose. However, significant differences were observed among them by ELISA. In conclusion, this study established the best conditions to produce MenW conjugate bulks and showed the efficacy of the obtained conjugate bulk in induce a good immune response in mice. Further experiments will need to be done to scale up the conjugation reaction and then allow the use of this conjugate in clinical trials.

Sphingolipid Inhibitors as an Alternative to Treat Candidiasis Caused by Fluconazole-Resistant Strains.

Candida species are fungal pathogens known to cause a wide spectrum of diseases, and Candida albicans and Candida glabrata are the most common associated with invasive infections. A concerning aspect of invasive candidiasis is the emergence of resistant isolates, especially those highly resistant to fluconazole, the first choice of treatment for these infections. Fungal sphingolipids have been considered a potential target for new therapeutic approaches and some inhibitors have already been tested against pathogenic fungi. The present study therefore aimed to evaluate the action of two sphingolipid synthesis inhibitors, aureobasidin A and myriocin, against different C. albicans and C. glabrata strains, including clinical isolates resistant to fluconazole. Susceptibility tests of aureobasidin A and myriocin were performed using CLSI protocols, and their interaction with fluconazole was evaluated by a checkerboard protocol. All Candida strains tested were sensitive to both inhibitors. Regarding the evaluation of drug interaction, both aureobasidin A and myriocin were synergic with fluconazole, demonstrating that sphingolipid synthesis inhibition could enhance the effect of fluconazole. Thus, these results suggest that sphingolipid inhibitors in conjunction with fluconazole could be useful for treating candidiasis cases, especially those caused by fluconazole resistant isolates.

Miltefosine Against Scedosporium and Lomentospora Species: Antifungal Activity and Its Effects on Fungal Cells.

Scedosporium and Lomentospora species are filamentous fungi responsible for a wide range of infections in humans and are frequently associated with cystic fibrosis and immunocompromising conditions. Because they are usually resistant to many antifungal drugs available in clinical settings, studies of alternative targets in fungal cells and therapeutic approaches are necessary. In the present work, we evaluated the in vitro antifungal activity of miltefosine against Scedosporium and Lomentospora species and how this phospholipid analogue affects the fungal cell. Miltefosine inhibited different Scedosporium and Lomentospora species at 2-4 µg/ml and reduced biofilm formation. The loss of membrane integrity in Scedosporium aurantiacum caused by miltefosine was demonstrated by leakage of intracellular components and lipid raft disorganisation. The exogenous addition of glucosylceramide decreased the inhibitory activity of miltefosine. Reactive oxygen species production and mitochondrial activity were also affected by miltefosine, as well as the susceptibility to fluconazole, caspofungin and myoricin. The data obtained in the present study contribute to clarify the dynamics of the interaction between miltefosine and Scedosporium and Lomentospora cells, highlighting its potential use as new antifungal drug in the future.

Peptidogalactomannan from Histoplasma capsulatum yeast cell wall: role of the chemical structure in recognition and activation by peritoneal macrophages.

Histoplasma capsulatum is the causative agent of histoplasmosis, a systemic disease responsible for most reported causes of morbidity and mortality among immunosuppressed individuals. Peptidogalactomannan (pGM) was purified from the yeast cell wall of H. capsulatum isolated from bats, and its structure and involvement in modulating the host immune response were evaluated. Gas chromatography, methylation analysis, and two-dimensional nuclear magnetic resonance (2D-NMR) were used for the structural characterization of pGM. Methylation and 2D-NMR data revealed that pGM comprises a main chain containing α-D-Manp (1 → 6) residues substituted at O-2 by α-D-Manp (1 → 2)-linked side chains, non-reducing end units of α-D-Galf, or ß-D-Galp linked (1→ 6) to α-D-Manp side chains. The involvement of H. capsulatum pGM in antigenic reactivity and in interactions with macrophages was demonstrated by ELISA and phagocytosis assay, respectively. The importance of the carbohydrate and protein moieties of pGM in sera reactivity was evaluated. Periodate oxidation abolished much pGM antigenic reactivity, suggesting that the sugar moiety is the most immunogenic part of pGM. Reactivity slightly decreased in pGM treated with proteinase K, suggesting that the peptide moiety plays a minor role in pGM antigenicity. In vitro experiments suggested that pGM is involved in the phagocytosis of H. capsulatum yeast and induction of IL-10 and IFN-γ secretion by peritoneal macrophages from C57BL/6 mice. These findings demonstrated the role of pGM in the H. capsulatum-host interaction.

Influence of relevant cystic fibrosis bacteria on Scedosporium apiospermum and Scedosporium boydii growth and viability.

Cystic fibrosis (CF) causes a variety of symptoms in different organs, but the majority of the morbidity and mortality of CF is related with pulmonary conditions. Primary infections are usually bacterial, and when treated with antibiotics, yeast infections appear or become more evident. Studies show that different microorganisms can co-inhabit the same environment and the interactions could be synergistic or antagonistic. Using techniques including viable and non-viable cell-to-cell interactions, mixed culture in liquid, and solid media sharing or not the supernatant, this study has evaluated interactions between the fungal species Scedosporium apiospermum and Scedosporium boydii with the bacterial species Staphylococcus aureus, Pseudomonas aeruginosa, and Burkholderia cepacia. Cell-to-cell interactions in liquid medium showed that P. aeruginosa and B. cepacia were able to reduce fungal viability but only in the presence of alive bacteria. Interactions without cell contact using a semi-permeable membrane showed that all bacteria were able to inhibit both fungal growths/viabilities. Cell-free supernatants from bacterial growth reduced fungal viability in planktonic fungal cells as well as in some conditions for preformed fungal biomass. According to the chemical analysis of the bacterial supernatants, the predominant component is protein. In this work, we verified that bacterial cells and their metabolites, present in the supernatants, can play anti-S. apiospermum and anti-S. boydii roles on fungal growth and viability.

Peptidorhamanomannan: A surface fungal glycoconjugate from Scedosporium aurantiacum and Scedosporium minutisporum and its recognition by macrophages.

The genus Scedosporium is composed of clinically relevant fungal species, such as Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium boydii. Surface molecules have been described that play crucial roles in fungi-macrophage interaction, and many of them are pathogen-associated molecular patterns (PAMPs). The present study aims to characterize peptidoglycans obtained from Scedosporium aurantiacum and Scedosporium minutisporum, a clinical and an environmental isolate, respectively, and compare their roles in pathogen-host interaction. Both molecules were characterized as peptidorhamnomannans (PRMs), similar to what has been already described for other Scedosporium species. Rabbit immune sera obtained by injecting whole cells from each species recognized both fungal cells and purified PRMs, suggesting that a cross-reaction occur between both fungi. Immunofluorescent microscopy revealed that PRMs are exposed on fungal surface. Prior incubation of purified molecules with immune sera before adding to cells led to loss of fluorescent, indicating that PRM is a major molecule recognized by immune sera. Fungi-macrophage interaction revealed that S. aurantiacum is able to survive more inside phagocytic cells than S. minutisporum, and PRM from both fungi plays a role in phagocytosis when the purified molecule is pre-incubated with macrophage. In addition, PRM induce nitric oxide release by macrophages. Our data indicate that PRM is an important PAMP exposed on fungal surface with the potential of immune modulation.

In this work, peptidorhamnomannans from Scedosporium aurantiacum and Scedosporium minutisporum have been characterized. These molecules play important roles in phagocytosis and oxidative burst in peritoneal macrophages and are recognized by immune sera.

Glucosylceramide Plays a Role in Fungal Germination, Lipid Raft Organization and Biofilm Adhesion of the Pathogenic Fungus Scedosporium aurantiacum.

Infections caused by Scedosporium species present a wide range of clinical manifestations, from superficial to disseminated, especially in immunocompromised patients. Glucosylceramides (GlcCer) are glycosphingolipids found on the fungal cell surface and play an important role in growth and pathogenicity processes in different fungi. The present study aimed to evaluate the structure of GlcCer and its role during growth in two S. aurantiacum isolates. Purified GlcCer from both isolates were obtained and its chemical structure identified by mass spectrometry. Using ELISA and immunofluorescence techniques it was observed that germination and NaOH-treatment of conidia favor GlcCer exposure. Monoclonal anti-GlcCer antibody reduced germination when cultivated with the inhibitor of melanin synthesis tricyclazole and also reduced germ tube length of conidia, both cultivated or not with tricyclazole. It was also demonstrated that anti-GlcCer altered lipid rafts organization, as shown by using the fluorescent stain filipin, but did not affect the susceptibility of the cell surface to damaging agents. Anti-GlcCer reduced total biomass and viability in biofilms formed on polystyrene plates. In the presence of anti-GlcCer, germinated S. aurantiacum conidia and biofilms could not adhere to polystyrene with the same efficacy as control cells. These results highlight the relevance of GlcCer in growth processes of S. aurantiacum.

Peptidorhamnomannans From Scedosporium and Lomentospora Species Display Microbicidal Activity Against Bacteria Commonly Present in Cystic Fibrosis Patients.

Scedosporium and Lomentospora species are filamentous fungi that cause a wide range of infections in humans. They are usually found in the lungs of cystic fibrosis (CF) patients and are the second most frequent fungal genus after Aspergillus species. Several studies have been recently performed in order to understand how fungi and bacteria interact in CF lungs, since both can be isolated simultaneously from patients. In this context, many bacterial molecules were shown to inhibit fungal growth, but little is known about how fungi could interfere in bacterial development in CF lungs. Scedosporium and Lomentospora species present peptidorhamnomannans (PRMs) in their cell wall that play crucial roles in fungal adhesion and interaction with host epithelial cells and the immune system. The present study aimed to analyze whether PRMs extracted from Lomentospora prolificans, Scedosporium apiospermum, Scedosporium boydii, and Scedosporium aurantiacum block bacterial growth and biofilm formation in vitro. PRM from L. prolificans and S. boydii displayed the best bactericidal effect against methicillin resistant Staphylococcus aureus (MRSA), Burkholderia cepacia, and Escherichia coli, but not Pseudomonas aeruginosa, all of which are the most frequently found bacteria in CF lungs. In addition, biofilm formation was inhibited in all bacteria tested using PRMs at minimal inhibitory concentration (MIC). These results suggest that PRMs from the Scedosporium and Lomentospora surface seem to play an important role in Scedosporium colonization in CF patients, helping to clarify how these pathogens interact to each other in CF lungs.

Antiviral activity of glucosylceramides isolated from Fusarium oxysporum against Tobacco mosaic virus infection.

Natural elicitors derived from pathogenic microorganisms represent an ecologic strategy to achieve resistance in plants against diseases. Glucosylceramides (GlcCer) are classified as neutral glycosphingolipids. GlcCer were isolated and purified from Fusarium oxysporum mycelium. F. oxysporum is a plant pathogenic fungus, abundant in soil and causing severe losses in economically important crops such as corn, tobacco, banana, cotton and passion fruit. In this study we evaluate the capacity of GlcCer in inducing resistance in N. tabacum cv Xanthi plants against Tobacco mosaic virus (TMV). Spraying tobacco plants with GlcCer before virus infection reduced the incidence of necrotic lesions caused by TMV. In addition, plants already infected with the virus showed a reduction in hypersensitive response (HR) lesions after GlcCer treatment, suggesting an antiviral effect of GlcCer. Our investigations showed that GlcCer stimulates the early accumulation of H2O2 and superoxide radicals. In addition, the expression of PR-1 (pathogenesis-related 1, with suggested antifungal action), PR-2 (ß-1,3-glucanase), PR-3 (Chitinase), PR-5 (Osmotin), PAL (Phenylalanine ammonia-lyase), LOX (Lipoxygenase) and POX (Peroxidase) genes was highly induced after treatment of tobacco plants with GlcCer and induction levels remained high throughout a period of 6 to 120 hours. Our experiments demonstrate that GlcCer induces resistance in tobacco plants against infection by TMV.