Molecular and cellular responses of the pathogenic fungus Lomentospora prolificans to the antifungal drug voriconazole

The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses.This work has been supported by grants (GIU15/36 and UFI11/25) from the UPV/EHU. AP was supported by a predoctoral fellowship from the UPV/EHU, and IB and AA were supported by predoctoral fellowships from the Basque Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A First Evaluation of the Usefulness of Kudzu Starch in Cultural Heritage Restoration

In recent times, the use of natural and harmless products for the environment and restorer is taking place in the field of Cultural Heritage restoration. In this sense, wheat, rice and corn starches as adhesives, have suitable characteristics without toxicity risks. A new starch in this field, is the Kudzu, an almost pure compound (99.5% starch) that is processed by a natural way from a plant called Pueraria lobata. This is a preliminary study of the potential use of Kudzu starch for the restoration of Cultural Heritage, focusing, firstly, in its capacity as adhesive through a comparative evaluation with common starches. The accelerated aging process carried out proved that Kudzu ensures optimal chromatic behaviour. On the other hand, the main problem in starch paste is the biological colonization. The daidzein, a natural antimicrobial compound implicit in Kudzu starch, confirmed the resistance to microorganism in this preliminary approach. The evaluation of the adhesive capacity, and the reversibility of the starches, suggest that Kudzu starch is a valid adhesive in the field of paper restoration. Thus, the potential of this starch in the conservation of Cultural Heritage is evidenced and its use as cleaner, resistance to biological colonization and consolidant is promising.This work has been supported by the MADyLIN project (Grant No. BIA2017-87063-P) funded by the Spanish Agency for Research AEI (MINEICO/FEDER-UE). The author wishes to acknowledge professional support of the Interdisciplinary Thematic Platform from CSIC Open Heritage: Research and Society (PTI-PAIS). Technical and human support provided Macroconduct, Mesostructure and Nanotechnology by SGIker (UPV/EHU, MICINN, GV/EJ, ERDF and ESF) are also gratefully acknowledge

Cyclophilin and enolase are the most prevalent conidial antigens of Lomentospora prolificans recognized by healthy human salivary IgA and cross-react with Aspergillus fumigatus

Purpose: The study of the immunocompetent airways immune response may provide important information to improve the therapeutic efficacy against Lomentospora (Scedosporium) prolificans. So, this study aimed to identify the most prevalent conidial antigens of this multiresistant fungus recognized by healthy human salivary immunoglobulin A, and to study their expression and cross-reactivity with other fungal species. Experimental design: Twenty saliva from immunocompetent donors were used to detect and identify the immunoreactive proteins by 2D immunoblotting and LC-MS/MS. Moreover, anti-Aspergillus antibodies were purified to study their cross-reactivity. Results: Ten proteins of L. prolificans conidia showed reactivity with more than 50% of the saliva samples. Among them, cyclophilin and enolase were the most prevalent antigens recognized by 85 and 80% of the samples, respectively. These enzymes were also identified on the cell wall surface of L. prolificans and on the immunomes of Scedosporium apiospermum and Scedosporium aurantiacum. Additionally, they showed cross-reactivity with the most common pathogenic filamentous fungus Aspergillus fumigatus. Conclusion and clinical relevance: These results show that the immunocompetent immune response might offer a pan-fungal recognition of conserved antigens such as enolase and cyclophilins, making them potential candidates for study as therapeutic targets.This work has been partially supported by several grants (EHUA13/14, UFI11/25, GIU15/36) from the University of the Basque Country (UPV/EHU). I. B. and A. A. were supported by a fellowship from the Basque Government, and Aize Pellon was supported by a fellowship from the UPV/EHU

The Host Immune Response to Scedosporium/Lomentospora

Infections caused by the opportunistic pathogens Scedosporium/Lomentospora are on the rise. This causes problems in the clinic due to the difficulty in diagnosing and treating them. This review collates information published on immune response against these fungi, since an understanding of the mechanisms involved is of great interest in developing more effective strategies against them. Scedosporium/Lomentospora cell wall components, including peptidorhamnomannans (PRMs), α-glucans and glucosylceramides, are important immune response activators following their recognition by TLR2, TLR4 and Dectin-1 and through receptors that are yet unknown. After recognition, cytokine synthesis and antifungal activity of different phagocytes and epithelial cells is species-specific, highlighting the poor response by microglial cells against L. prolificans. Moreover, a great number of Scedosporium/Lomentospora antigens have been identified, most notably catalase, PRM and Hsp70 for their potential medical applicability. Against host immune response, these fungi contain evasion mechanisms, inducing host non-protective response, masking fungal molecular patterns, destructing host defense proteins and decreasing oxidative killing. In conclusion, although many advances have been made, many aspects remain to be elucidated and more research is necessary to shed light on the immune response to Scedosporium/Lomentospora.This research was funded by the Basque Government, grant number IT1362-19. L.M.-S., M.A., and L.A.-F. were funded by the Basque Government

Nitrogen, Iron, and Zinc Acquisition: Key Nutrients to Aspergillus fumigatus Virulence

Aspergillus fumigatus is a ubiquitous soil decomposer and an opportunistic pathogen that is characterized by its large metabolic machinery for acquiring nutrients from media. Lately, an ever-increasing number of genes involved in fungal nutrition has been associated with its virulence. Of these, nitrogen, iron, and zinc metabolism-related genes are particularly noteworthy, since 78% of them have a direct implication in virulence. In this review, we describe the sensing, uptake and regulation process of the acquisition of these nutrients, the connections between pathways and the virulence-implicated genes. Nevertheless, only 40% of the genes mentioned in this review have been assayed for roles in virulence, leaving a wide field of knowledge that remains uncertain and might offer new therapeutic and diagnostic targets.This research was funded by the Basque Government: grant number IT1362-19. U.P.-C. and S.C.-S. received a predoctoral fellowship from the University of the Basque Country and Basque Government, respectively

ELISA Test for the Serological Detection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for Scedosporium/Lomentospora, four different Scedosporium boydii protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the Scedosporium/Lomentospora-infected from Aspergillus-infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect Scedosporium spp. and Lomentospora prolificans with very high sensitivity and specificity, 86%-100% and 93%-99%, respectively, depending on the cut-off value chosen (four values were proposed A(450nm)= 0.5837, A(450nm)= 0.6042, A(450nm)= 0.6404, and A(450nm)= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presenceThis research was funded by the Basque Government, grant number IT1362-19. IB, LM-S, and LA-F received a predoctoral fellowship from the Basque Government. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the result

The monoclonal antibody Ca37, developed against Candida albicans alcohol dehydrogenase, inhibits the yeast in vitro and in vivo

Candida albicans is a commensal yeast able to cause life threatening invasive infections particularly in immunocompromised patients. Despite the availability of antifungal treatments, mortality rates are still unacceptably high and drug resistance is increasing. We, therefore, generated the Ca37 monoclonal antibody against the C. albicans alcohol dehydrogenase (Adh) 1. Our data showed that Ca37 was able to detect C. albicans cells, and it bound to Adh1 in yeast and Adh2 in hyphae among the cell wall-associated proteins. Moreover, Ca37 was able to inhibit candidal growth following 18h incubation time and reduced the minimal inhibitory concentration of amphotericin B or fluconazole when used in combination with those antifungals. In addition, the antibody prolonged the survival of C. albicans infected-Galleria mellonella larvae, when C. albicans was exposed to antibody prior to inoculating G. mellonella or by direct application as a therapeutic agent on infected larvae. In conclusion, the Ca37 monoclonal antibody proved to be effective against C. albicans, both in vitro and in vivo, and to act together with antifungal drugs, suggesting Adh proteins could be interesting therapeutic targets against this pathogen.Technical and human support provided by the Proteomics Core Facility-SGIker at the UPV/EHU is gratefully acknowledged. We thank the member of the Chartered of Linguists, No 022913 for improving the English in the manuscript. This work was supported by Basque Government (Grant IT1362-19). AA, IB and LMS have received a predoctoral Grant from Basque Government and LAF from UPV/EH

Dot Immunobinding Assay for the Rapid Serodetection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

The detection of Scedosporium/Lomentospora is still based on non-standardized low-sensitivity culture procedures. This fact is particularly worrying in patients with cystic fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because a poor and delayed diagnosis can worsen the prognosis of the disease. To contribute to the discovery of new diagnostic strategies, a rapid serological dot immunobinding assay (DIA) that allows the detection of serum IgG against Scedosporium/Lomentospora in less than 15 min was developed. A crude protein extract from the conidia and hyphae of Scedosporium boydii was employed as a fungal antigen. The DIA was evaluated using 303 CF serum samples (162 patients) grouped according to the detection of Scedosporium/Lomentospora in the respiratory sample by culture, obtaining a sensitivity and specificity of 90.48% and 79.30%, respectively; positive and negative predictive values of 54.81% and 96.77%, and an efficiency of 81.72%. The clinical factors associated with the results were also studied using a univariate and a multivariate analysis, which showed that Scedosporium/Lomentospora positive sputum, elevated anti-Aspergillus serum IgG and chronic Pseudomonas aeruginosa infection were significantly associated with a positive result in DIA, while Staphylococcus aureus positive sputum showed a negative association. In conclusion, the test developed can offer a complementary, rapid, simple and sensitive method to contribute to the diagnosis of Scedosporium/Lomentospora in patients with CF.This research was funded by the Basque Government, grant numbers IT1362-19 and IT1657-22. L.M-S and M.A have received a predoctoral grant from the Basque Government and L.A-F from the University of the Basque Country (UPV/EHU)

Aspergillus fumigatus Fumagillin Contributes to Host Cell Damage

The activity of fumagillin, a mycotoxin produced by Aspergillus fumigatus, has not been studied in depth. In this study, we used a commercial fumagillin on cultures of two cell types (A549 pneumocytes and RAW 264.7 macrophages). This toxin joins its target, MetAP2 protein, inside cells and, as a result, significantly reduces the electron chain activity, the migration, and the proliferation ability on the A549 cells, or affects the viability and proliferation ability of the RAW 264.7 macrophages. However, the toxin stimulates the germination and double branch hypha production of fungal cultures, pointing out an intrinsic resistant mechanism to fumagillin of fungal strains. In this study, we also used a fumagillin non-producer A. fumigatus strain (∆fmaA) as well as its complemented strain (∆fmaA::fmaA) and we tested the fumagillin secretion of the fungal strains using an Ultra High-Performance Liquid Chromatography (UHPLC) method. Furthermore, fumagillin seems to protect the fungus against phagocytosis in vitro, and during in vivo studies using infection of immunosuppressed mice, a lower fungal burden in the lungs of mice infected with the ∆fmaA mutant was demonstrated.This research was funded by the Basque Government: grant number IT1362-19. X.G. and S.C.-S. received a Ph.D. fellowship from the Basque Government; and U.P.-C. from the University of the Basque Country

Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.This work was supported by two SAIOTEK grants (S-PE09UN41 and S-PE11UN070) and a consolidated research group IT343/10 of Basque Government,two grants from the University of the Basque Country (UE 03/04 and UE 08/14) and a UFI Grant (UFI11/25) from the University of the Basque Country UPV/EHU. Andoni Ramirez-Garcia was supported by a fellowship from the Government of the Basque Countr