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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.

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.

[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.

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.

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.

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.

The Añana Salt Valley in Spain is an active continental solar saltern formed 220 million years ago. To date, no fungal genomic studies of continental salterns have been published, although DNA metabarcoding has recently expanded researchers’ ability to study microbial community structures. Accordingly, the aim of this present study was to evaluate fungal diversity using the internal transcribed spacer (ITS) metabarcoding at different locations along the saltern (springs, ponds, and groundwater) to describe the fungal community of this saline environment. A total of 380 fungal genera were detected. The ubiquity of Saccharomyces was observed in the saltern, although other halotolerant and halophilic fungi like Wallemia, Cladosporium, and Trimmatostroma were also detected. Most of the fungi observed in the saltern were saprotrophs. The fungal distribution appeared to be influenced by surrounding conditions, such as the plant and soil contact, cereal fields, and vineyards of this agricultural region.

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.

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 presence

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.

The high mortality rates of Lomentospora prolificans infections are due, above all, to the tendency of the fungus to infect weakened hosts, late diagnosis and a lack of effective therapeutic treatments. To identify proteins of significance for diagnosis, therapy or prophylaxis, immunoproteomics-based studies are especially important. Consequently, in this study murine disseminated infections were carried out using L. prolificans, Scedosporium aurantiacum, Scedosporium boydii and Aspergillus fumigatus, and their sera used to identify the most immunoreactive proteins of L. prolificans total extract and secreted proteins. The results showed that L. prolificans was the most virulent species and its infections were characterized by a high fungal load in several organs, including the brain. The proteomics study showed a high cross-reactivity between Scedosporium/Lomentospora species, but not with A. fumigatus. Among the antigens identified were, proteasomal ubiquitin receptor, carboxypeptidase, Vps28, HAD-like hydrolase, GH16, cerato-platanin and a protein of unknown function that showed no or low homology with humans. Finally, Hsp70 deserves a special mention as it was the main antigen recognized by Scedosporium/Lomentospora species in both secretome and total extract. In conclusion, this study identifies antigens of L. prolificans that can be considered as potential candidates for use in diagnosis and as therapeutic targets and the production of vaccines.

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.

Many studies have analyzed relationships between microorganisms and cancer, demonstrating that microorganisms are able to prevent the onset of cancer and, others to provoke it. Specifically, more and more scientific articles are publishing on microorganisms, linking them to the creation, implementation and dispersion of cancer. In fact, it is estimated that microorganisms cause 17.8% of all cancers. The cancer-causing viral capability is the most studied and, in consequence, many different viral mechanisms that can cause cancer have been described. The International Cancer Re-search Institute has categorized eight viruses for the first time as "carcinogenic to hu-mans", including a human papillomavirus, two herpesvirus and two hepatitis viruses. Regarding bacteria, among cancerous agents, Helicobacter pylori is the most studied in relation to stomach cancer. In addition, many other bacteria, such as Salmonella typhi, Chlamydia pneumoniae and Streptococcus bovis, have been directly related to cancer. Although relatively little research on the effect of fungi on cancer has been investi-gated, some of the toxins produced by these microorganisms have been shown to cause cancer. In addition, some mechanism for the generation and spread of cancer have been described in Candida albicans. Studies to date have shown the influence of microor-ganisms on the development and promotion of cancer. For this reason, to face cancer in the next future, deepen into the relationship between cancer and microorganisms will be essential; Ikerketa askok mikroorganismoen eta minbizien arteko erlazioak aztertu dituzte, eta erakutsi dute mikroorganismo batzuek minbiziaren agerpena saihesten dutela eta beste batzuek, aldiz, minbizia eragin dezaketela. Hain zuzen ere, gero eta artikulu zientifiko gehiago argitaratzen ari dira mikroorganismoak minbiziaren sortzearekin, ezarpenarekin eta sakabanaketarekin erlazionatuz. Izan ere, mikroorganismoek minbizi guztien % 17,8 eragiten dutela estimatu da.

Lomentospora (Scedosporium) prolificans is an opportunistic pathogen capable of causing invasive infections in immunocompromised patients. The fungus is able to disseminate via the bloodstream finally arriving at the central nervous system producing neurological symptoms and, in many cases, patient death. In this context, microglial cells, which are the resident immune cells in the central nervous system, may play an important role in these infections. However, this aspect of anti-L. prolificans immunity has been poorly researched to date. Thus, the interactions and activity of microglial cells against L. prolificans were analysed, and the results show that there was a remarkable impairment in their performance regarding phagocytosis, the development of oxidative burst, and in the production of pro-inflammatory cytokines, compared with macrophages. Interestingly, L. prolificans displays great growth also when challenged with immune cells, even when inside them. We also proved that microglial phagocytosis of the fungus is highly dependent on mannose receptor and especially on dectin-1. Taken together, these data provide evidence for an impaired microglial response against L. prolificans and contribute to understanding the pathobiology of its neurotropism.

Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.

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.

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.

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.

[EN] Background: Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism of PCR products (PCR-RFLP) are extensively used molecular biology techniques. An exercise for the design and simulation of PCR and PCR-RFLP experiments will be a useful educational tool. Findings: An online PCR and PCR-RFLP exercise has been create that requires users to find the target genes,compare them, design primers, search for restriction endonucleases, and finally to simulate the experiment. Each user of the service is randomly assigned a gene from Escherichia coli; to complete the exercise, users must design an experiment capable of distinguishing among E. coli strains. By applying the experimental procedure to all completely sequenced E. coli, a basic understanding of strain comparison and clustering can also be acquired. Comparison of results obtained in different experiments is also very instructive. Conclusions: The exercise is freely available at http://insilico.ehu.es/edu.