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.

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.

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.

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.

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.

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.

Scedosporium Cell Wall: From Carbohydrate-Containing Structures to Host-Pathogen Interactions.

Scedosporium species are filamentous fungi usually found in sewage and soil from human-impacted areas. They cause a wide range of diseases in humans, from superficial infections, such as mycetoma, to invasive and disseminated cases, especially associated in immunocompromised patients. Scedosporium species are also related to lung colonization in individuals presenting cystic fibrosis and are considered one of the most frequent fungal pathogens associated to this pathology. Scedosporium cell wall contains glycosylated molecules involved in important biological events related to virulence and pathogenicity and represents a significant source of antigens. Polysaccharides, peptidopolysaccharides, O-linked oligosaccharides and glycosphingolipids have been identified on the Scedosporium surface. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-MS, and 1H and 13C nuclear magnetic resonance. Peptidorhamnnomannans are common cell wall components among Scedosporium species. Comparing different species, minor structural differences in the carbohydrate portions were detected which could be useful to understand variations in virulence observed among the species. N- and O-linked peptidorhamnomannans are major pathogen-associated molecular patterns and, along with α-glucans, play important roles in triggering host innate immunity. Glycosphingolipids, such as glucosylceramides, have highly conserved structures in Scedosporium species and are crucial for fungal growth and virulence. The present review presents current knowledge on structural and functional aspects of Scedosporium glycoconjugates that are relevant for understanding pathogenicity mechanisms and could contribute to the design of new agents capable of inhibiting growth and differentiation of Scedosporium species. Other cell components such as melanin and ectophosphatases will be also included.

Sphingolipid biosynthetic pathway is crucial for growth, biofilm formation and membrane integrity of Scedosporium boydii.

Aim: Glycosphingolipids are conserved lipids displaying a variety of functions in fungal cells, such as determination of cell polarity and virulence. They have been considered as potent targets for new antifungal drugs. The present work aimed to test two inhibitors, myriocin and DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, in Scedosporium boydii, a pathogenic fungus which causes a wide range of disease. Materials & methods: Mass spectrometry, microscopy and cell biology approaches showed that treatment with both inhibitors led to defects in fungal growth and membrane integrity, and caused an increased susceptibility to the current antifungal agents. Conclusion: These data demonstrate the antifungal potential of drugs inhibiting sphingolipid biosynthesis, as well as the usefulness of sphingolipids as promising targets for the development of new therapeutic options.