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.

Peptidorhamnomannan from Lomentospora prolificans modulates the inflammatory response in macrophages infected with Candida albicans.

BACKGROUND: Peptidorhamnomannan is a glycoconjugate that consists of a peptide chain substituted by O- and N-linked glycans, present on the cell surface of Lomentospora prolificans, a saprophytic fungus which is widely distributed in regions with temperate climates. O-linked oligosaccharides from peptidorhamnomannan isolated from Lomentospora prolificans conidia are recognized by macrophages mediating macrophage - conidia interaction. In this work, peptidorhamnomannan was isolated from L. prolificans mycelium cell wall and its role in macrophage - Candida albicans interaction was evaluated. RESULTS: Purified peptidorhamnomannan inhibits the reactivity of rabbit immune sera to mycelial and conidia forms of L. prolificans, indicating that this glycoconjugate is exposed on the fungal surface and can mediate interaction with host immune cells. We demonstrated that peptidorhamnomannan leads to TNF-α production in J774 macrophages for 1, 2 and 3 h of incubation, suggesting that this glycoconjugate may have a beneficial role in the response to fungal infections. In order to confirm this possibility, the effect of peptidorhamnomannan on the macrophage - C. albicans interaction was evaluated. Macrophages treated with peptidorhamnomannan led to a lower fungal survival, suggesting that peptidorhamnomannan induces an increased fungicidal activity in macrophages. Furthermore, TNF-α levels were measured in supernatants after macrophage - C. albicans interaction for 1, 2 and 3 h. Peptidorhamnomannan treatment led to a higher TNF-α production at the beginning of the interaction. However, the release of TNF-α was not maintained after 1 h of incubation. Besides, peptidorhamnomannan did not show any inhibitory or fungicidal effect in C. albicans when used at 100 µg/ml but it was able to kill C. albicans at a concentration of 400 µg/ml. CONCLUSION: We suggest that peptidorhamnomannan acts as a molecular pattern on the invading pathogen, promotes TNF-α production and, thus, increases macrophage fungicidal activity against Candida albicans.

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.

Monohexosylceramides from Rhizopus Species Isolated from Brazilian Caatinga: Chemical Characterization and Evaluation of Their Anti-Biofilm and Antibacterial Activities.

Monohexosylceramides (CMHs) are highly conserved fungal glycosphingolipids playing a role in several cellular processes such as growth, differentiation and morphological transition. In this study, we report the isolation, purification and chemical characterization of CMHs from Rhizopus stolonifer and R. microspores. Using positive ion mode ESI-MS, two major ion species were observed at m/z 750 and m/z 766, respectively. Both ion species consisted of a glucose/galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to a hydroxylated C16:0 fatty acid. The antimicrobial activity of CMH was evaluated against Gram positive and Gram negative bacteria using the agar diffusion assay. CMH from both Rhizopus species inhibited the growth of Bacillus terrae, Micrococcus luteus (M. luteus) and Pseudomonas stutzeri (P. stutzeri) with a MIC50 of 6.25, 6.25 and 3.13 mg/mL, respectively. The bactericidal effect was detected only for M. luteus and P. stutzeri, with MBC values of 25 and 6.25 mg/mL, respectively. Furthermore, the action of CMH on the biofilm produced by methicillin-resistant Staphylococcus aureus (MRSA) was analyzed using 12.5 and 25 mg/mL of CMH from R. microsporus. Total biofilm biomass, biofilm matrix and viability of the cells that form the biofilm structure were evaluated. CMH from R. microsporus was able to inhibit the MRSA biofilm formation in all parameters tested.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Glucosylceramides From Lomentospora prolificans Induce a Differential Production of Cytokines and Increases the Microbicidal Activity of Macrophages.

Lomentospora prolificans is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions, such as involvement in fungal growth and morphological transitions in several fungi. The elucidation of the primary structure of the fungal surface glycoconjugates could contribute for the understanding of the mechanisms of pathogenicity. In this work, GlcCer species were isolated from mycelium and conidia forms of L. prolificans and their chemical structures were elucidated by mass spectrometry (ESI-MS). GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N-2-hydroxyhexadecanoyl-1-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize L. prolificans GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, in vivo assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of L. prolificans conidia by peritoneal macrophages. These results indicate that GlcCer species from L. prolificans are a potent immune response activator.