A novel SPE-UHPLC-DAD method for the determination of fumagillin produced by Aspergillus fumigatus in cell culture media

[EN]Fumagillin is a biomolecule produced by Aspergillus fumigatus that is gaining relevance due to its connection with invasive aspergillosis. The determination of this molecule might help to understand the propagation of this disease and study its use as a potential biomarker. In spite of the interest of fumagillin in microbiological research, no quantitative method has been developed so far for its determination in cell culture media. In this work, the first validated method for the quantitative analysis of fumagillin in RPMI-1640 is presented. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled the validation criteria established by EMA and FDA guidelines for bioanalysis (selectivity, carry over, linearity, accuracy, precision, dilution integrity and stability) and offers a limit of quantitation (25 μg·L−1) suitable for its intended use. Indeed, the method was satisfactorily applied to the quantification of the fumagillin produced by three strains of Aspergillus fumigatus with different toxin production capacity.This research was funded by University of the Basque Country (UPV/EHU) (project GIU19/068 and project COLAB20/11) and by Basque Government (grant number IT1362-19). XGP was funded by Basque Government

Fumagilina mikotoxinak aspergilosi inbaditzailearen garapenean duen rola aztertzeko SPE-UHPLC-DAD metodo analitikoa

Invasive aspergillosis caused by Aspergillus fumigatus is a threat for immunocompromised patients. According to recent studies, fumagillin, a mycotoxin produced by this fungus, has been associated with the propagation of the disease. Therefore, this molecule might help to understand the mechanisms of this disease and to study the use of fumagillin as a potential biomarker of invasive aspergillosis. In spite of the relevance of fumagillin analysis in microbiological research, no quanti-tative method has been developed so far for its determination in cell culture media. Here, we present the first validated method for the quantitative analysis of fumagillin in RPMI-1640. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled all the validation criteria established by EMA (European Medicine Agency) and FDA (Food and Drug Administration) guidelines for bioanalysis. Finally, the method was satisfactorily applied to the quantification of the fumagillin produced by different strains of Aspergillus fumigatus and it was observed that they had a different micotoxin production capacity.; Aspergillus fumigatus onddoak sortutako aspergilosi inbaditzailea mehatxua da immunoeskasia duten gai-xoentzat. Azkeneko ikerketa batzuen arabera, fumagilinak, onddoak sortutako mikotoxinak, gaixotasunaren hedapenarekin zerikusia duela ikusi da. Hori dela eta, konposatu honen determinazioa lagungarria izan daiteke bai gaixotasunaren mekanismoak hobeto ulertzeko eta baita aspergilosi inbaditzailearen biomarkatzaile gisa erabili ahal izateko ere. Ikerketa mikro-biologikoetan fumagilinaren analisiak garrantzia izan arren, oraindik ez da haren determinaziorako metodo kuantitatiborik garatu zelula-hazkuntzako inguruneetan. Beraz, lan honetan fumagilinaren determinazio kuantitatiborako lehenengo metodo analitikoa balidatu da RPMI-1640 zelula-hazkuntzako ingurunean. Laginaren tratamendua fase solidoko erauzketarekin egin da, anioi trukatzaile sendoak diren modu mistoko kartutxoak erabiliz. Horrela, egon daitezkeen interferentziak modu eraginkorrean ezabatu dira, eta % 83 ± 7ko berreskurapena lortu da. Analisia fotodiodo detektagailuari akoplaturiko bereizmen oso altuko likido kromatografia erabiliz egin da 336 nm-ko uhin-luzeran. Horrela, metodoak EMA (Europako Medikamentuen Agentzia) eta FDA (Elikagai eta Sendagaien Administrazioa) agentziek balidazio bioanalitikoetarako zehazten dituzten parametro guztien onartze-irizpideak betetzen dituela egiaztatu da. Gero, metodoa A. fumigatus-en lau andui analizatzeko aplikatu da, eta bakoitzak mikotoxinaren kantitate desberdina ekoizteko gaitasuna daukala ikusi da

Study of antifungal agent caspofungin adsorption to laboratory materials

[EN] Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.The authors thank University of the Basque Country (UPV/EHU) (Project GIU19/068 and Project COLAB20/11) and Basque Government (grant number IT1362-19) for financial support. B. Uribe thanks UPV/EHU for the pre-doctoral fellowship in co-supervision with the University of Bordeaux. X. Guruceaga thanks the Basque Government for his predoctoral grant

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

Flexible multiplex PCR to detect SARS-CoV-2, coronavirus OC43 and influenza A virus in nasopharyngeal swab samples

Introduction Quantitative reverse transcription PCR (RT-qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits. Methods and results In this study, combinations of four primers/probe sets were used to construct a flexible RT-qPCR assay which is capable of discriminating between SARS-CoV-2 and the seasonal human coronavirus HCoV-OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed. Conclusions This study provides an efficient method for the simultaneous detection of SARS-CoV-2, HCoV-OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results. Significance and impact of the study The multiplex RT-qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.This research was funded by Basque Government, grants numbers 2020333042 and IT1362‐19. LM‐S have received a predoctoral Grant from Basque Government

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

Study of the Aspergillus fumigatus host-pathogen interaction using transcriptomic techniques and mutant strains generation

251 p.Aspergillus fumigatus is a filamentous fungus capable to cause a wide range of diseases depending on the immune status of the patient who breath its conidia. The most dangerous disease caused by this fungus is known as invasive aspergilosis and its diagnosis and treatment is frequently delayed and useless. Therefore, describing new virulence factors that could serve as new diagnostic or therapeutic targets is crucial. In this PhD thesis, we have designed a whole genome transcriptomic study in different infection conditions with the aim to define which Aspergillus fumigatus genes are differentially expressed during the infective process. The project is divided in three related chapters in which we have defined genes that could be important for the establishment and development of the Aspergillus fumigatus infection. Specifically, we have found that during the first 96 hours of an intranasal infection, the genes related with the biosynthesis of fumagillin/pseurotina, several genes that codify proteases (dppIV, dppV, aspf1 or aspf5), chitinase (chiB1) as well as some genes related with pyomelanin biosynthesis (hppD and maiA) are overexpressed. Furthermore, we have described that fumagillin could be an important virulence factor for the fungus because this mycotoxin causes cell death and inhibition of the cell metabolism and proliferation. In the same way, the toxin is a protective factor against phagocytosis and plays an important role during the infection process. Finally, using CRISPR-Cas9 technology we have proved that Aspergillus fumigatus maiA gene is involved in the fungal cell wall synthesis, the correct hyphae development and the fungal virulence

Identification and functional characterization of the putative members of the CTDK-1 kinase complex as regulators of growth and development in Aspergillus nidulans and Aspergillus fumigatus

Asexual spores are the main vehicle used by fungi to disperse to new niches. The Eurotiomycete Aspergillus nidulans is the main reference for the study of the genetic/molecular control of asexual development. In this species, Flb proteins control the expression of the master gene brlA, and thus, loss-of-function mutations in flb (upstream developmental activation [UDA]) genes block brlA transcription and, consequently, the production of conidiophores, the structures bearing asexual spores known as conidia. However, the aconidial phenotype of specific flb mutants, such as that of the ΔflbB strain, is reverted under salt-stress conditions. Previously, we generated a collection of second-site mutants of ΔflbB unable to conidiate on culture medium supplemented with NaH2PO4 (0.65 M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype and characterized the role of the putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex in A. nidulans and Aspergillus fumigatus. FlpA, Stk47, and FlpB are necessary (i) for timely germination, (ii) in the transition from metulae to phialides (the cells generating conidia) during conidiophore development, and (iii) for the development of sexual structures (cleistothecia) in A. nidulans. The three proteins are nuclear, and the nucleoplasmic localization of Stk47 depends on the activity of FlpA, which correlates with the retention of Stk47 by FlpA in pull-down assays. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control. Identification of a mutation in flpA as inhibitor of conidiation in A. nidulans and functional characterization of FlpA, Stk47 and FlpB as putative members of the C-terminal domain kinase complex CTDK-1 in A. nidulans and A. fumigatus.Work at O.E.'s lab has been supported by Universidad del País Vasco (UPV)/Euskal Herriko Unibertsitatea (EHU) (GIU19/014) and the Basque Government (PIBA-PUE PIBA_2020_1_0032; Elkartek KK-2021/43 and KK-2022/00107; and GIC IT1662-22). Work at CIB Margarita Salas-CSIC has been supported by Ministerio de Ciencia, Innovaciόn y Universidades/Spanish Agencia Estatal de Investigación (RTI2018-094263-B-100, to E.A.E.). A.O. held a Margarita Salas grant (MARSA21/69), funded by Next-Generation EU, at the UPV/EHU. A.F. was a degree student with a collaboration grant by the Spanish Ministry of Education (21/19070). Z.A. was a master thesis student at O.E.'s lab, held an Ikertalent Fellowship funded by the Basque Government (PIF21/003) at the Basque Culinary Center, and is now a PhD student at O.E.'s lab with funds of the KK-2022/00107 project. Work at J.R.F.'s lab has been supported by National Institutes of Health grants R01-AI158442 and R01-AI143197. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.This research was funded by the Basque Government, grant number IT1362-19. X.G. and U.P.-C. were funded by the Basque Government and the UPV/EHU, respectively

The Aspergillus fumigatus maiA gene contributes to cell wall homeostasis and fungal virulence

In this study, two distinct in vitro infection models of Aspergillus fumigatus, using murine macrophages (RAW264.7) and human lung epithelial cells (A549), were employed to identify the genes important for fungal adaptation during infection. Transcriptomic analyses of co-incubated A. fumigatus uncovered 140 fungal genes up-regulated in common between both models that, when compared with a previously published in vivo transcriptomic study, allowed the identification of 13 genes consistently up-regulated in all three infection conditions. Among them, the maiA gene, responsible for a critical step in the L-phenylalanine degradation pathway, was identified. Disruption of maiA resulted in a mutant strain unable to complete the Phe degradation pathway, leading to an excessive production of pyomelanin when this amino acid served as the sole carbon source. Moreover, the disruption mutant exhibited noticeable cell wall abnormalities, with reduced levels of β-glucans within the cell wall but did not show lack of chitin or mannans. The maiA-1 mutant strain induced reduced inflammation in primary macrophages and displayed significantly lower virulence in a neutropenic mouse model of infection. This is the first study linking the A. fumigatus maiA gene to fungal cell wall homeostasis and virulence