Identificación inmunoproteómica de antígenos de Lomentospora prolificans con interés diagnóstico y terapéutico.

264 p.Este estudio inmunoproteómico ha permitido la identificación de antígenos de Lomentospora prolificans de relevancia para el diagnóstico y el tratamiento. Concretamente, se ha descrito una elevada prevalencia de reactividad de las IgA salivares de humanos sanos frente a la ciclofilina y la enolasa del hongo, lo que, junto a su localización en la superficie celular, hacen de estas enzimas unas candidatas potenciales a ser estudiadas como dianas terapéuticas o para el desarrollo de vacunas. Además, se ha demostrado la reactividad cruzada de estas enzimas con A. fumigatus, lo que puede ser de interés para el desarrollo de tratamientos eficaces frente a ambos géneros. Por otro lado, se ha demostrado que L. prolificans es más virulento que Scedosporium spp. y A. fumigatus. El estudio de los sueros obtenidos de infecciones murinas permitió la identificación de los antígenos inmunodominantes de L. prolificans. Algunas de las proteínas identificadas, como el receptor proteasomal de ubiquitina, CPY, Vps28, hidrolasa tipo HAD, GH16, Hp jhhlp_006787, la cerato-platanina y la Hsp70 pueden ser interesantes como dianas diagnósticas y terapéutic

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

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

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

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

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)

Identificación inmunoproteómica de antígenos de Lomentospora prolificans con interés diagnóstico y terapéutico.

264 p.Este estudio inmunoproteómico ha permitido la identificación de antígenos de Lomentospora prolificans de relevancia para el diagnóstico y el tratamiento. Concretamente, se ha descrito una elevada prevalencia de reactividad de las IgA salivares de humanos sanos frente a la ciclofilina y la enolasa del hongo, lo que, junto a su localización en la superficie celular, hacen de estas enzimas unas candidatas potenciales a ser estudiadas como dianas terapéuticas o para el desarrollo de vacunas. Además, se ha demostrado la reactividad cruzada de estas enzimas con A. fumigatus, lo que puede ser de interés para el desarrollo de tratamientos eficaces frente a ambos géneros. Por otro lado, se ha demostrado que L. prolificans es más virulento que Scedosporium spp. y A. fumigatus. El estudio de los sueros obtenidos de infecciones murinas permitió la identificación de los antígenos inmunodominantes de L. prolificans. Algunas de las proteínas identificadas, como el receptor proteasomal de ubiquitina, CPY, Vps28, hidrolasa tipo HAD, GH16, Hp jhhlp_006787, la cerato-platanina y la Hsp70 pueden ser interesantes como dianas diagnósticas y terapéutic

Mikroorganismoek minbizia eragin dezakete?

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. Minbizia sortzeko birusen gaitasuna da gehien ikertu dena eta, ondorioz, minbizia sor dezaketen mekanismo desberdin asko deskribatu dira. Minbizia Ikertzeko Nazioarteko Agentziak zortzi birus 1. mailako "gizakiontzat kartzinogeno"-tzat sailkatu ditu; haien artean, giza papiloma birusa, bi herpesbirus eta bi hepatitisaren birus aurkitzen dira. Bakterioei dagokienez, minbizi-eragileen artean, Helicobacter pylori da gehien ikertu dena urdaileko minbiziarekin erlazionatuta. Baina honetaz gain, beste hainbat bakterio, hala nola Salmonella typhi, Chlamydia pneumoniae eta Streptococcus bovis minbiziarekin zuzenki erlazionatu dira. Onddoek daukaten minbiziarekiko erlazioa oso gutxi ikertu den arren, mikroorganismo hauek sortutako toxina batzuek minbizia eragin dezaketela frogatu da. Horrez gain, Candida albicans onddoak minbiziaren sorrera eta hedapena eragin dezakeen hainbat mekanismo deskribatu dira. Orain arte egindako ikerketek mikroorganismoek minbiziaren garapenean eta sustapenean daukaten eragina agerrarazi dute. Hori dela eta, etorkizunean minbiziari aurre egiteko, minbiziaren eta mikroorganismoen arteko erlazioan sakontzea ezinbestekoa da

Study of Humoral Responses against Lomentospora/Scedosporium spp. and Aspergillus fumigatus to Identify L. prolificans Antigens of Interest for Diagnosis and Treatment

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.This research was funded by the Basque Government, grant number IT1362-19. I.B., L.M.S. and L.A.F. received a predoctoral fellowship from the Basque Government