Distinct proteomes and allergen profiles appear across the life-cycle stages of Alternaria alternata.

BACKGROUND: Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus. OBJECTIVE: We sought to determine differences between spore and hyphae proteomes and how allergens are distributed in Aalternata. METHODS: Data-independent acquisition mass spectrometry was used to quantitatively compare the proteomes of asexual spores (nongerminating and germinating) with vegetative hyphae. RESULTS: We identified 4515 proteins in nongerminating spores, germinating spores, and hyphae; most known allergens are more abundant in nongerminating spores. On comparing significant protein fold-change differences between nongerminating spores and hyphae, we found that 174 proteins were upregulated in nongerminating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. On comparing nongerminating and germinating spores, 25 proteins were found to be upregulated in nongerminating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media. CONCLUSIONS: Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in nongerminating spores.

Genetic Polymorphisms of P2RX7 but Not of ADORA2A Are Associated with the Severity of SARS-CoV-2 Infection.

SARS-CoV-2 infection ranges from mild to severe presentations, according to the intensity of the aberrant inflammatory response. Purinergic receptors dually control the inflammatory response: while adenosine A2A receptors (A2ARs) are anti-inflammatory, ATP P2X7 receptors (P2X7Rs) exert pro-inflammatory effects. The aim of this study was to assess if there were differences in allelic and genotypic frequencies of a loss-of-function SNP of ADORA2A (rs2298383) and a gain-of-function single nucleotide polymorphism (SNP) of P2RX7 (rs208294) in the severity of SARS-CoV-2-associated infection. Fifty-five individuals were enrolled and categorized according to the severity of the infection. Endpoint genotyping was performed in blood cells to screen for both SNPs. The TT genotype (vs. CT + CC) and the T allele (vs. C allele) of P2RX7 SNP were found to be associated with more severe forms of COVID-19, whereas the association between ADORA2A SNP and the severity of infection was not significantly different. The T allele of P2RX7 SNP was more frequent in people with more than one comorbidity and with cardiovascular conditions and was associated with colorectal cancer. Our findings suggest a more prominent role of P2X7R rather than of A2AR polymorphisms in SARS-CoV-2 infection, although larger population-based studies should be performed to validate our conclusions.

Azorean Black Tea (Camellia sinensis) Antidermatophytic and Fungicidal Properties.

The treatment of dermatophytoses, the most common human fungal infections, requires new alternatives. The aim of this study was to determine the antidermatophytic activity of the aqueous Azorean Black Tea extract (ABT), together with an approach to the mechanisms of action. The phytochemical analysis of ABT extract was performed by HPLC. The dermatophytes susceptibility was assessed using a broth microdilution assay; potential synergies with terbinafine and griseofulvin were evaluated by the checkerboard assay. The mechanism of action was appraised by the quantification of the fungal cell wall chitin and ß-1,3-glucan, and by membrane ergosterol. The presence of ultrastructural modifications was studied by Transmission Electron Microscopy (TEM). The ABT extract contained organic and phenolic acids, flavonoids, theaflavins and alkaloids. It showed an antidermatophytic effect, with MIC values of 250 µg/mL for Trichophyton mentagrophytes, 125 µg/mL for Trichophyton rubrum and 500 µg/mL for Microsporum canis; at these concentrations, the extract was fungicidal. An additive effect of ABT in association to terbinafine on these three dermatophytes was observed. The ABT extract caused a significant reduction in ß-1,3-glucan content, indicating the synthesis of this cell wall component as a possible target. The present study identifies the antidermatophytic activity of the ABT and highlights its potential to improve the effectiveness of conventional topical treatment currently used for the management of skin or mucosal fungal infections.

Filamentous Fungi Extracellular Vesicles.

Since the first description of extracellular vesicles in a filamentous fungus, Alternaria infectoria, data has been gathered showing the importance of EVs in the interaction of filamentous fungi with the environment and with the animal and plant hosts. In Aspergillus spp. it was described paracrine effects over host cells, namely regulating the immune response; in phytopathogens, it was described the importance of EVs in infection structures. The study of the filamentous fungi EVs associated with cargos indicates important roles in the breakdown of substrates and the remodeling of the cell wall. Nevertheless, the information about filamentous fungi EVs is still scarce and the biogenesis and release deserve further study.

Clustering of adenosine A2B receptors with ectonucleotidases in caveolin-rich lipid rafts underlies immunomodulation by Leishmania amazonensis.

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

Feature extraction from MRI ADC images for brain tumor classification using machine learning techniques.

BACKGROUND: Diffusion-weighted (DW) imaging is a well-recognized magnetic resonance imaging (MRI) technique that is being routinely used in brain examinations in modern clinical radiology practices. This study focuses on extracting demographic and texture features from MRI Apparent Diffusion Coefficient (ADC) images of human brain tumors, identifying the distribution patterns of each feature and applying Machine Learning (ML) techniques to differentiate malignant from benign brain tumors. METHODS: This prospective study was carried out using 1599 labeled MRI brain ADC image slices, 995 malignant, 604 benign from 195 patients who were radiologically diagnosed and histopathologically confirmed as brain tumor patients. The demographics, mean pixel values, skewness, kurtosis, features of Grey Level Co-occurrence Matrix (GLCM), mean, variance, energy, entropy, contrast, homogeneity, correlation, prominence and shade, were extracted from MRI ADC images of each patient. At the feature selection phase, the validity of the extracted features were measured using ANOVA f-test. Then, these features were used as input to several Machine Learning classification algorithms and the respective models were assessed. RESULTS: According to the results of ANOVA f-test feature selection process, two attributes: skewness (3.34) and GLCM homogeneity (3.45) scored the lowest ANOVA f-test scores. Therefore, both features were excluded in continuation of the experiment. From the different tested ML algorithms, the Random Forest classifier was chosen to build the final ML model, since it presented the highest accuracy. The final model was able to predict malignant and benign neoplasms with an 90.41% accuracy after the hyper parameter tuning process. CONCLUSIONS: This study concludes that the above mentioned features (except skewness and GLCM homogeneity) are informative to identify and differentiate malignant from benign brain tumors. Moreover, they enable the development of a high-performance ML model that has the ability to assist in the decision-making steps of brain tumor diagnosis process, prior to attempting invasive diagnostic procedures, such as brain biopsies.

Retrospective study of group A Streptococcus oropharyngeal infection diagnosis using a rapid antigenic detection test in a paediatric population from the central region of Portugal.

Group A Streptococcus (GAS) is one of the most important agents of oropharyngeal infection. To avoid unnecessary antibiotic prescription, it is recommended the confirmation of GAS infection in pharyngeal swabs using culture or rapid antigen detection test (RADT). This study aimed to retrospectively analyse the incidence of GAS oropharyngeal infection, detected by RADT, in a paediatric population in the Centre of Portugal. Data was collected from the database of the Paediatric Hospital Emergency Department (ED) regarding patients admitted with symptoms suggesting acute pharyngitis, from January 2013 to December 2018, in a total of 18,304 cases. Among these, 130 clinical files were searched for symptoms, complications and additional visits to the ED. The results showed an average GAS infection prevalence of 33%, with seasonal variation. In preschool children, especially in patients less than 3 years old, where the guidelines do not routinely encourage RADT, GAS tonsillitis assumed an unexpected importance, with 731 positive tests in a total of 3128 cases. Scarlatiniform rash and oral cavity petechiae had significant correlation with streptococcal aetiology (p < 0.05). The statistical analysis also showed that different signs and symptoms assume different weights depending on the age group of the patient. The main conclusion is that the incidence of GAS infection in the studied population is higher than generally described in preschool children, suggesting the need for a more cautious approach to children under 3 years presenting acute pharyngitis, and that RADT in this age group would contribute to a decrease in the number of unnoticed cases.

Evaluation of overtime phenotypic variation of yeasts in chronic vulvovaginal candidosis cases.

Chronic vulvovaginal candidosis results either from reinfection or from the ability of Candida spp. to persist in the vulva and/or vagina. Persistence is usually associated with increased antifungal (mainly azoles) resistance rates, which can explain treatment failure, and/or increased expression of virulence factors by Candida spp. The aim of this study was to assess the mechanisms leading to Candida spp persistence, by studying sequential isolates from women with chronic vulvovaginal candidosis, focusing on strains genotypes, azole resistance, and ability to form biofilms along the period of clinical evaluation. The strains were identified at species level by automated analysis of biochemical profiles and molecular typing evaluated by polymorphic DNA analysis. The capacity to form biofilm was assessed with a microtiter plate assay. Fluconazole susceptibility was determined by the microdilution broth assay at both pH 7 (following the recommended guideline) and pH 4.5 (as representative of vaginal pH). We studied samples from 17 clinically recurrent cases. In 53% of the chronic cases there were two or more isolates that had a phylogenetic relationship while the remaining (47%) were caused by different species. In those cases where related strains were involved in recurrence, we verified an increase in MIC at pH 7 and also an increased capacity to form biofilms over time. Significant correlation between these two parameters was observed only in cases caused by C. glabrata, evidencing the importance of these two factors to enhance persistence in the vaginal mucosa for this particular species.

Chronic vulvovaginal candidosis (VVC) affects millions of women worldwide. We found that persistence of the same Candida strain on the vaginal mucosa does not account for the great majority of VVC cases. Moreover, modulation of biofilm formation and azole resistance overtime was investigated.

Recurrent vulvovaginal Candida spp isolates phenotypically express less virulence traits.

OBJECTIVE: Vulvovaginal candidosis (VVC) is a condition that impacts the quality of life of women worldwide. At least 5-8% of all VVC cases re-occur. Recurrent vulvovaginal candidosis (RVVC) can be defined as the occurrence of a VVC episode at least four times per year. The reasons for recurrence to occur are poorly understood. This work aims to identify key phenotypic traits associated with RVVC Candida spp. isolates that might be used to plan strategies to control RVVC. METHODS: The capacity to form biofilms (with the microtitration plate assay), to develop germinative tube in the presence of fetal bovine serum and to produce phospholipase (in the egg-yolk plate assay) was assessed for a collection of Candida spp. isolates obtained from 17 women diagnosed with RVVC and 16 women with non-recurrent VVC (VVC). The differences obtained regarding the proportion of isolates expressing each virulence factor was assessed by statistical analysis (χ2). RESULTS: We found that C. albicans isolates had a higher ability to form germinative tubes than RVVC isolates (29% vs 4%, p < 0.05). In addition, the ability of Candida spp. isolates to form biofilm (63% vs 51%) and to produce phospholipase (13% vs 11%) was also higher, though not statistically different (p > 0.05). CONCLUSIONS: We conclude that biofilm formation and phenotypic-switching associated with germinative tube production are particularly important C. albicans virulence factors for acute, sporadic VVC cases.

Evaluation of a novel dog animal model for peri-implant disease: clinical, radiographic, microbiological and histological assessment.

OBJECTIVE: To assess longitudinal peri-implant tissue evaluation in a plaque compromised ligature free dog model, clinically, radiographically, microbiologically and histologically. MATERIALS AND METHODS: Six beagle mandibular premolars and first molars were extracted. Plaque accumulated for 16 weeks. Two implants were placed per hemi-mandible. For 17 weeks, control implants (CI) in one hemi-mandible were brushed daily; test implants (TI) in the other were not. These parameters were then assessed: clinically, probing depth (PD), bleeding-on-probing (BOP), presence of plaque (PP) and clinical attachment level (CAL); radiographically, marginal bone level; microbiologically, counts for Streptococcus spp., Fusobacterium spp., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia and total bacterial load. At week 17, histomorphometric analysis was performed (MM-ISH (mucosal margin-implant shoulder); ISH-fBIC (implant shoulder-first bone-to-implant contact); MM-aJE (mucosal margin-apical area junctional epithelium); MM-aINF (mucosal margin-apical limit of the inflammatory infiltrate); %INF (percentage of inflammatory infiltrate)). RESULTS: At week 17, TI had significant increased PD, BOP, PP and CAL versus baseline. All clinical variables presented intergroup differences. There was no intergroup difference for radiographic bone loss (p > 0.05). Total bacteria, Fusobacterium spp., A. actinomycetemcomitans and P. gingivalis had intergroup differences. There was no statistically significant intergroup difference for ISH-fBIC. CONCLUSIONS: Longitudinal microbiology evaluation detected a shift period. Final intergroup microbiological differences were the basis of W17 clinical intergroup differences, with higher values in TI. Microbiological and clinical changes detected in peri-implant tissues were compatible with onset of peri-implant disease. Despite histological inflammatory intergroup difference, no histological or radiographic intergroup bone loss was detected. CLINICAL RELEVANCE: This study set-up describes a valuable method for generating "true" early peri-implant defects without mechanical trauma.

Intestinal Microbial and Metabolic Profiling of Mice Fed with High-Glucose and High-Fructose Diets.

Increased sugar intake is implicated in Type-2 diabetes and fatty liver disease; however, the mechanisms through which glucose and fructose promote these conditions are unclear. We hypothesize that alterations in intestinal metabolite and microbiota profiles specific to each monosaccharide are involved. Two groups of six adult C57BL/6 mice were fed for 10-weeks with diets with glucose (G) or fructose (F) as sole carbohydrates, and a third group was fed with a normal chow carbohydrate mixture (N). Fecal metabolites were profiled by nuclear magnetic resonance (NMR) and microbial composition by real-time polymerase chain reaction (qPCR). Although N, G and F mice exhibited similar weight gains (with slight slower gains for F) and glucose tolerance, multivariate analysis of NMR data indicated that F mice were separated from N and G, with decreased butyrate and glutamate and increased fructose, succinate, taurine, tyrosine, and xylose. The different sugar diets also resulted in distinct intestinal microbiota profiles. That associated with fructose seemed to hold more potential to induce host metabolic disturbances compared to glucose, mainly by promoting bile acid deconjugation and taurine release and compromising intestinal barrier integrity. This may reflect the noted nonquantitative intestinal fructose absorption hence increasing its availability for microbial metabolism, a subject for further investigation.

Type 1 diabetes in children is not a predisposing factor for oral yeast colonization.

Type 1 diabetes mellitus (T1D) is considered a risk factor associated with oral yeast infections. The aim of this study was to evaluate the yeast oral carriage (in saliva and mucosal surface) of children with T1D and potential relation with host factors, particularly the subset of CD4+ T cells. Yeasts were quantified and identified in stimulated saliva and in cheek mucosal swabs of 133 diabetic T1D and 72 healthy control subjects. Salivary lymphocytes were quantified using flow cytometry. The presence of yeasts in the oral cavity (60% of total patients) was not affected by diabetes, metabolic control, duration of the disease, salivary flow rate or saliva buffer capacity, by age, sex, place of residence, number of daily meals, consumption of sweets or frequency of tooth brushing. Candida albicans was the most prevalent yeast species, but a higher number of yeast species was isolated in nondiabetics. T1D children with HbA1c ≤ 7.5 (metabolically controlled) presented higher number of CD4+ T salivary subsets when compared with the other groups of children (non-diabetic and nonmetabolically controlled) and also presented the highest number of individuals without oral yeast colonization. In conclusion, T1D does not predisposes for increased oral yeast colonization and a higher number of salivary CD4+T cells seems to result in the absence of oral colonization by yeasts.

HIV1-viral protein R (Vpr) mutations: associated phenotypes and relevance for clinical pathologies.

Over the last 30 years, research into HIV has advanced the knowledge of virus genetics and the development of efficient therapeutic strategies. HIV-1 viral protein R (Vpr) is a specialized and multifunctional protein that plays important roles at multiple stages of the HIV-1 viral life cycle. This protein interacts with a number of cellular and viral proteins and with multiple activities including nuclear transport of the pre-integration complex (PIC) to the nucleus, transcriptional activation, cell cycle arrest at G2/M transition phase and induction of cell death via apoptosis. Specifically, Vpr has been shown to control many host cell functions through a variety of biological processes and by interaction with several cellular pathways. The different functions of Vpr may enhance viral replication and impair the immune system in HIV-1 infected patients. Importantly, functional defects induced by mutations in the Vpr protein correlate with slow disease progression of HIV-infected patients. Vpr is also associated with other concomitant pathologies developed by these patients, which may lead it to be considered as a potential novel therapeutic target. This review will focus on HIV-1 Vpr, mainly on the importance of its structural mutations on the progression of HIV infection, associated phenotypes and relevance for clinical pathologies. Copyright © 2016 John Wiley & Sons, Ltd.

Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans.

Human infection with Cryptococcus neoformans, a common fungal pathogen, follows deposition of yeast spores in the lung alveoli. The subsequent host-pathogen interaction can result in eradication, latency, or extrapulmonary dissemination. Successful control of C. neoformans infection is dependent on host macrophages, but macrophages display little ability to kill C. neoformans in vitro. Recently, we reported that ingestion of C. neoformans by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by C. neoformans toxicity. Infection of macrophages by C. neoformans was associated with alterations in protein translation rate and activation of several stress pathways, such as hypoxia-inducing factor-1-α, receptor-interacting protein 1, and apoptosis-inducing factor. Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated, depending on macrophage cell type, consistent with the nonspecific nature of C. neoformans virulence known to infect phylogenetically distant hosts. Our results indicate that C. neoformans infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen.

Triatoma maculata colonises urban domicilies in Boa Vista, Roraima, Brazil.

During a medical entomology course in Boa Vista, Roraima, colonies of Triatoma maculata closely associated with pigeon nests were observed in concrete air-conditioner box located on the external plastered and cemented walls of a modern brick-built apartment block. In only one eight-hole ceramic brick, located inside one air-conditioner box, 127 specimens of T. maculata were collected. T. maculata is a recognised vector of Trypanosoma cruzi in the surrounding area and its domiciliation increases the risk of Chagas disease transmission.

Different danger signals differently impact on microglial proliferation through alterations of ATP release and extracellular metabolism.

Microglia rely on their ability to proliferate in the brain parenchyma to sustain brain innate immunity and participate in the reaction to brain damage. We now studied the influence of different danger signals activating microglia, both internal (typified by glutamate, associated with brain damage) and external (using a bacterial lipopolysaccharide, LPS), on the proliferation of microglia cells. We found that LPS (100 ng/mL) increased, whereas glutamate (0.5 mM) decreased proliferation. Notably, LPS decreased whereas glutamate increased the extracellular levels of ATP. In contrast, LPS increased whereas glutamate decreased the extracellular catabolism of ATP into adenosine through ecto-nucleotidases and ecto-5'-nucleotidase. Finally, apyrase (degrades extracellular ATP) abrogated glutamate-induced inhibition of microglia proliferation; conversely, inhibitors of ecto-nucleotidases (ARL67156 or α,ß-methylene ADP) and adenosine deaminase (degrades extracellular adenosine) abrogated the LPS-induced increase of microglia proliferation, which was blocked by a selective A2A receptor antagonist, SCH58261 (50 nM). Overall, these results highlight the importance of the extracellular purinergic metabolism to format microglia proliferation and influence the spatio-temporal profile of neuroinflammation in different conditions of brain damage.

Activation of Melanin Synthesis in Alternaria infectoria by Antifungal Drugs.

The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.

Modulation of Alternaria infectoria cell wall chitin and glucan synthesis by cell wall synthase inhibitors.

The present work reports the effects of caspofungin, a ß-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ß-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ß-glucan synthase inhibitor against this fungus.

Characterization of Alternaria infectoria extracellular vesicles.

Many fungi use membrane vesicles to transport complex molecules across their cell walls. Like mammalian exosomes, fungal vesicles contain lipids, proteins, and polysaccharides, many of which are associated with virulence. Here we identify and characterize extracellular vesicles (EVs) in Alternaria infectoria, a ubiquitous, environmental filamentous fungus that is also an opportunistic human pathogen. Examination of the A. infectoria EVs revealed a morphology similar to that of vesicles described in other fungal species. Of note, proteomic analysis detected a reduced number of vesicle-associated proteins. There were two prevalent categories among the 20 identified proteins, including the polysaccharide metabolism group, probably related to plant host invasion or biosynthesis/degradation of cell wall components, and the nuclear proteins, especially DNA repair enzymes. We also found enzymes related to pigment synthesis, adhesion to the host cell, and trafficking of vesicles/organelles/molecules. This is the first time EV secretions have been identified in a filamentous fungus. We believe that these vesicles might have a role in virulence.