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Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii . Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus  Paracoccidioides  spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4 + and CD8 + T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4 + T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Silva, Taira, Cleare, Martins, Junqueira, Nosanchuk and Taborda.)

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I 2  = 90.7%; 87% CI = (84-89), P < 0.001] and specificity [I 2  = 89.2%; 93% CI = (92-95), P < 0.001] was observed. The performance of diagnostic tests was 0.93. Enzyme-linked immunosorbent assay was the main tool used, and the ConA-binding fraction antigen of the strain 1099-18 appears as a promising diagnostic biomarker candidate., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, the genetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein in Pichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, the highest yield of Hcp100 reached approximately 1.3 mg/l with > 90% purity in shake flasks using basal salt medium supplemented with casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samples using specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis (PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a pool of 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study provides preliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapid diagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic tool of PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patient survival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largely unknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostic tests. KEY POINTS: Few antigens are used as laboratory tools for the immunodiagnosis of histoplasmosis. P. pastoris was an excellent system for recombinant Hcp100 expression. Maximum expression levels of rHcp100 were achieved in BSM with 1% casamino acids. Dot blot assays with anti-rHcp100 antisera can be successfully used for diagnosing PHD.

Background: Despite all the efforts made up to now, the reasons that facilitate a protein becoming an allergen have not been elucidated yet. Alt a 1 protein is the major fungal allergen responsible for chronic asthma, but little is known about its immunological activity. Our main purpose was to investigate the ligand-dependent interactions of Alt a 1 in the human airway epithelium., Methods: Alt a 1 with and without its ligand (holo- and apo- forms) was incubated with the pulmonary epithelial monolayer model, Calu-3 cells. Allergen transport and cytokine production were measured. Pull-down and immunofluorescence assays were employed to identify the receptor of Alt a 1 using the epithelial cell model and mouse tissues. Receptor-allergen-ligand interactions were analyzed by computational modeling., Results: The holo-form could activate human monocytes, PBMCs, and polarized airway epithelial (Calu-3) cell lines. The allergen was also transported through the monolayer, without any alteration of the epithelial integrity (TEER). Alt a 1 also induced the production of proinflammatory IL8 and specific epithelial cytokines (IL33 and IL25) by Calu-3 cells. The interaction between epithelial cells and holo-Alt a 1 was found to be mediated by the SLC22A17 receptor, and its recognition of Alt a 1 was explained in structural terms., Conclusions: Our findings identified the Alt a 1 ligand as a central player in the interaction of the allergen with airway mucosa, shedding light into its potential role in the immunological response, while unveiling its potential as a new target for therapy intervention., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Cryptococcal meningitis has a high morbidity and mortality among AIDS population. Cryptococcal antigen (CrAg) detection is considered an independent predictor for meningitis and death. Since 2011, the World Health Organization recommends CrAg screening for people living with HIV/AIDS (PLHAs) with CD4 counts <100-200 cells/μl. Its implementation is still limited in low-middle-income countries. We aimed to estimate the prevalence and predictors of CrAg positivity in PLHAs. We also evaluated outcomes among those who were CrAg-positive., Methods: Prospective cohort conducted at an infectious diseases hospital, in Brazil. Adults with CD4 <200 cells/μl, without previous cryptococcal disease and regardless of symptoms, were enrolled from 2015 to 2018. CrAg tests were performed by LFA. Lumbar puncture was done in CrAg+ individuals and pre-emptive therapy was offered for those without meningitis., Results: Of 214 individuals recruited, 88% were antiretroviral experienced, of which only 11.6% with viral suppression. Overall, CrAg prevalence was 7.9% (95% CI, 4.7-12.4). In CD4 ≤100 cells/μl group it was 7.5% (95% CI, 4.1-12.6) and 9.1% (95% CI, 3.4-19.0) in the group with CD4 101 to 199 cells/μl (p = 0.17). Prevalence in asymptomatic subjects was 5.3% (95% CI, 1.4-13.1). One among 17 CrAg+ participants had documented meningoencephalitis and no subclinical meningitis was detected. Adherence to pre-emptive treatment was 68.7% (11/16). There were no statistically significant differences in sociodemographic, clinical or laboratory characteristics to predict CrAg positivity. No case of cryptococcal disease was diagnosed among CrAg + subjects, followed by a median of 12 months., Conclusions: CrAg screening for severely immunosuppressed PLHAs in Brazil yielded a prevalence of 7.9%. No difference was found in the prevalence of CrAg stratified by CD4 values (CD4 <100 versus CD4 101-199 cells/μl). No clinical nor laboratory factors predicted CrAg positivity, corroborating the need for the implementation of universal CrAg screening for PLHAs with CD4 <200 cells/μl in similar settings., Competing Interests: The authors have declared that no competing interests exist.

Alternaria is a major outdoor allergen. Immunotherapy with Alternaria extracts has been documented to be effective in the sensitized patients. However, Alternaria extracts are notoriously difficult to standardize. Our aim is to screen the B cell mimotopes of Alternaria and to evaluate the therapeutic effects of B cell mimotope peptides on a BALB/c mouse model of Alternaria allergy. After a human sera pool from Alternaria monosensitized patients was established, B cell mimotopes were screened by a phage-displayed random heptamer peptide library that was identified via mixed Alternaria -specific IgE in the sera pool. B cell mimotopes with phage as a carrier were used to perform immunotherapy in an Alternaria allergy mouse model. Serological Ab levels, lung histology, and cytokine profiles were compared in the mimotope immunotherapy group, natural extract immunotherapy group, irrelevant phage control group, Alternaria -sensitized model group, and saline-blank group. Two mimotopes (MISTSRK and QKRNTIT) presented high binding ability with the sera of the Alternaria -allergic patients and mice and, therefore, were selected for immunotherapy in the mouse model. Compared with irrelevant phage control, model, and natural extract immunotherapy group, mimotope immunotherapy group significantly reduced serum IgE levels, inflammatory cells infiltration in the lung tissue, and IL-4 levels in bronchoalveolar lavage fluid, whereas serum IgG1 and IFN-γ levels in bronchoalveolar lavage fluid were increased. Our results indicate that B cell mimotopes of Alternaria alleviates allergic response in a mouse model and have potential as novel therapeutic agents for IgE-mediated Alternaria -allergic diseases., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Bmtutl-519 is an isoform of the Bombyx Turtle protein and a member of the immunoglobulin superfamily (IgSF). The relative expression level of Bmtutl-519 was significantly upregulated when BmN cells were infected by Nosema bombycis. The subcellular localization of Bmtutl-519 was studied using an indirect immunoinfluscent assay (IFA), Co-localization assay, Western blotting, and enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bmtutl-519 expression plasmid. The results indicate that Bmtutl-519 is distributed in both the cytoplasm and the cell membrane of BmN cells. Bmtutl-519 may be involved in the infection process of N. bombycis as a cell surface receptor or regulatory factor. Interaction analysis of Bmtutl-519 with NbSWP26, a spore wall protein of N. bombycis involved in host cell adherence and infection, showed that the C-terminal heparin-binding motif (HBM) of NbSWP26 mediates the interaction between these two proteins. Mutation of the NbSWP26 HBM at K208G, K209G, K210G, and K213G led to a loss of the ability to bind the Bmtutl-519 protein. Spore adherence and infection assays showed that Bmtutl-519 enhances the binding ability of N. bombycis to the host cell surface, but this did not enhance host cell infection by N. bombycis. In contrast, the sustained high expression of Bmtutl-519 in BmN cells inhibited the proliferation of N. bombycis spores., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Pathologic diagnosis remains the gold standard for final diagnosis of acute invasive fungal sinusitis (AIFS); however, other less invasive tests could suggest the presence of AIFS in at-risk populations where early diagnosis is crucial. Serum galactomannan Aspergillus antigen has been shown to correlate with a diagnosis of invasive pulmonary aspergillosis; however, it has not adequately been evaluated in regard to AIFS. The objective of this study is to evaluate the statistical relevance of galactomannan in predicting diagnosis of AIFS., Methods: This study was a retrospective review of pathologic records using Co-Path from 2006 to 2017, incorporating 2 separate searches with designated criteria identifying patients who received pathologic evaluation for invasive fungal sinusitis. Electronic medical records were subsequently reviewed. After exclusions isolating at-risk populations and removing duplications, 78 cases were reviewed using the indicated search criteria. Of these, 38 met further criteria of having had both pathologic evaluation and galactomannan analysis. Statistical variables were assessed, as well as all-cause mortality. Peak and closest galactomannan levels were evaluated., Results: Overall, galactomannan had a sensitivity of 44.8% (95% confidence interval [CI], 26.5% to 64.3%), specificity of 100% (95% CI, 66.4% to 100%), positive predictive value of 100% (95% CI, 74.3% to 100%), and negative predictive value of 36% (95% CI, 18.0% to 57.5%). No significant association was observed in galactomannan status and mortality in this patient population., Conclusion: Positive serum galactomannan can be an indication of AIFS in patients with a high clinical suspicion. In our study, a positive galactomannan always correlated with a positive pathologic diagnosis. However, given its low sensitivity, one must use caution in relying on galactomannan as a screening tool in diagnosis of AIFS., (© 2018 ARS-AAOA, LLC.)

Pulmonary aspergillosis is a severe infectious disease caused by some members of the Aspergillus genus, that affects immunocompetent as well as immunocompromised patients. Among the different disease forms, Invasive Aspergillosis is the one causing the highest mortality, mainly, although not exclusively, affecting neutropenic patients. This genus is very well known by humans, since different sectors like pharmaceutical or food industry have taken advantage of the biological activity of some molecules synthetized by the fungus, known as secondary metabolites, including statins, antibiotics, fermentative compounds or colorants among others. However, during infection, in response to a hostile host environment, the fungal secondary metabolism is activated, producing different virulence factors to increase its survival chances. Some of these factors also contribute to fungal dissemination and invasion of adjacent and distant organs. Among the different secondary metabolites produced by Aspergillus spp. Gliotoxin (GT) is the best known and better characterized virulence factor. It is able to generate reactive oxygen species (ROS) due to the disulfide bridge present in its structure. It also presents immunosuppressive activity related with its ability to kill mammalian cells and/or inactivate critical immune signaling pathways like NFkB. In this comprehensive review, we will briefly give an overview of the lung immune response against Aspergillus as a preface to analyse the effect of different secondary metabolites on the host immune response, with a special attention to GT. We will discuss the results reported in the literature on the context of the animal models employed to analyse the role of GT as virulence factor, which is expected to greatly depend on the immune status of the host: why should you hide when nobody is seeking for you? Finally, GT immunosuppressive activity will be related with different human diseases predisposing to invasive aspergillosis in order to have a global view on the potential of GT to be used as a target to treat IA.

Introduction: Cryptococcal meningitis (CM) is an opportunistic fungal disease with a high mortality among HIV-positive patients with severe immunosuppression (CD4 count <100 cells/μl). Reflexed screening for cryptococcal antigen (CrAg) in remnant blood samples was initially piloted at selected CD4 testing laboratories of the National Health Laboratory Service (NHLS) prior to the implementation of a national screening programme using a lateral flow assay (LFA) (IMMY, Norman, OK, USA). The aim of this study was to assess CrAg positivity nationally, per province and district in combination with the percentage of CD4 samples tested with a CD4 count <100 cells/μl to identify areas with advanced HIV/CrAg disease burden., Methods: CrAg and CD4 laboratory result data were extracted from the NHLS corporate data warehouse. Monthly test volumes were used to assess CrAg test volumes and coverage, while bubble charts were used to display the relationship between CD4 <100 cells/μl, CrAg positivity and number of positive CrAg samples by district. ArcGIS software was used to spatially report CrAg positivity., Results: CrAg screening coverage was stable at around 96% after November 2016. Samples with a CD4 <100 cell/μl and CrAg positivity were also stable over the study period at 10% and ~5% respectively. The highest CrAg positivity was reported for the Kwa-Zulu Natal province (7.3%), which also had the lowest percentage of samples with a CD4 <100 cells/μl (7.2%). Uthungulu and Umkhanyakude districts had the highest CrAg positivity (9.3% and 8.9% respectively). Ethekwini and Johannesburg Metro districts contributed to 22% of the total number of CrAg-positive samples tested across South Africa for the period reported., Conclusion: Existing CD4 testing services were used to rapidly scale up CrAg reflex testing in South Africa. Districts with advanced HIV and CrAg disease burden were identified that need further investigation of patient management interventions., Competing Interests: The authors have declared that no competing interests exist.

The walls of both, yeast and mycelial cells of Candida albicans possess a species-specific antigen that is recognized by a monoclonal antibody (MAb 3H8). This antigen can be extracted in the form of a very high Mr complex, close or over 106 Da, by treatment, with β-1,3-glucanase, β mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall β-1,3 glucans, and to proteins by disulfide bonds, but not to β-1,6 glucans. Through its sensitivity to trypsin and different deglycosylation procedures, it was concluded that the epitope is associated to a glycoprotein containing N-glycosidic, but not O-glycosidic mannan moieties. By means of electrophoresis in polycrylamide gradient gels, followed by mass spectrometric analysis, the epitope was pinpointed to a very high MW complex containing Agglutinin-Like Sequence (ALS) family proteins, and other cytoplasmic, membrane and secreted proteins. The components of this complex are bound by unknown covalent bonds. The material extracted with β mercaptoethanol or dilute alkali appeared under the electron microscope as large aggregates in the form of spheroidal and mostly web-like structures of large sizes. These, and additional data, suggest that this protein complex may constitute an important part of the basic glycoprotein structure of C. albicans. The possibility that similar complexes exist in the wall of other fungi is an attractive, although yet untested possibility.

Aspergillus fumigatus and multiple other Aspergillus species cause a wide range of lung infections, collectively termed aspergillosis. Aspergilli are ubiquitous in environment with healthy immune systems routinely eliminating inhaled conidia, however, Aspergilli can become an opportunistic pathogen in immune-compromised patients. The aspergillosis mortality rate and emergence of drug-resistance reveals an urgent need to identify novel targets. Secreted and cell membrane proteins play a critical role in fungal-host interactions and pathogenesis. Using a computational pipeline integrating data from high-throughput experiments and bioinformatic predictions, we have identified secreted and cell membrane proteins in ten Aspergillus species known to cause aspergillosis. Small secreted and effector-like proteins similar to agents of fungal-plant pathogenesis were also identified within each secretome. A comparison with humans revealed that at least 70% of Aspergillus secretomes have no sequence similarity with the human proteome. An analysis of antigenic qualities of Aspergillus proteins revealed that the secretome is significantly more antigenic than cell membrane proteins or the complete proteome. Finally, overlaying an expression dataset, four A. fumigatus proteins upregulated during infection and with available structures, were found to be structurally similar to known drug target proteins in other organisms, and were able to dock in silico with the respective drug.

Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days after challenge with wild-type A. fumigatus QC5096, AFMP1 knockdown mutant, AFMP2 knockdown mutant and AFMP3 knockdown mutant and 28 days after challenge with AFMP4 knockdown mutant (P<.0001). Only 11% of mice died 30 days after challenge with AFMP1-4 knockdown mutant (P<.0001). For mice challenge with AFMP1-4 knockdown mutant, lower abundance of fungal elements was observed in brains, kidneys, and spleens compared to mice challenge with QC5096 at day 4 post-infection. Fungal counts in brains of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.01 and P<.05). Fungal counts in kidneys of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.001 and P<.001) and those of mice challenge with QC5096 were significantly higher than those challenge with AFMP4 knockdown mutant (P<.05). There is no difference among the survival rates of wild-type A. fumigatus, AFMP4 knockdown mutant and AFMP1-4 knockdown mutant, suggesting that Mp1p homologues in A. fumigatus do not mediate its virulence via improving its survival in macrophage as in the case in T. marneffei. Afmp1p, Afmp2p, Afmp3p, and Afmp4p in combination are important virulence factors of A. fumigatus.

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Apoptosis is considered an escape mechanism from the host immune system for the fungus Paracoccidioides spp, and it serves as a vehicle for entry into macrophages without stimulating microbicidal activities. Recently, gp43 of P. brasiliensis was demonstrated to be involved in this process. Therefore, as a new therapeutic alternative, it is very important to study compounds that could reduce the modulation of the induction of apoptosis caused by this fungus. Decyl gallate (G14) is a known antifungal compound, and we decided to investigate its anti-apoptotic properties. Our results demonstrate that G14 was effective against apoptosis induced by gp43, as observed in epithelial cells, and led to a reduction in DNA damage, Bak down-regulation and Bcl-2 up-regulation. Together, these data show that G14 presents promising anti-apoptotic activity., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Diagnosis of central nervous system cryptococcosis relies on a spectrum of methods but has improved with lateral flow diagnostic assays that detect capsular polysaccharide antigens of Cryptococcus. Here, we present the case of an HIV-infected African-American man with cryptococcal meningoencephalitis caused by a strain producing little or no capsule., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose of Review: Cryptococcal infections are an important cause of morbidity and mortality in solid organ transplant patients. Here, we review the microbiology, epidemiology, clinical course, treatment, and outcomes of Cryptococcus in solid organ transplant recipients., Recent Findings: We identify the unique findings in solid organ transplant patients when compared to other immunocompromised patients such as those with HIV. We also describe our experience and outcomes with regard to solid organ transplant patients who do not have positive fungal cultures, but cryptococcal antigen positivity and concern for cryptococcal disease., Summary: Our review will highlight the importance of these new diagnostic techniques in those with Cryptococcus and solid organ transplant, which will be the subject of new research.

Here, we report a patient developing a postoperative peritoneal infection by Aspergillus fumigatus. While galactomannan serum levels were negative throughout the time course, galactomannan levels in peritoneal fluids yielded high results. Serological testing of peritoneal fluids for fungal antigens might be a useful and easily applicable tool to support diagnosis of intraabdominal aspergillosis, which represents a rare type of invasive fungal infection.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of recombinant antigens has been shown to improve both the sensitivity and the standardization of the serological diagnosis of Farmer's lung disease (FLD). The aim of this study was to complete the panel of recombinant antigens available for FLD serodiagnosis with antigens of Lichtheimia corymbifera, known to be involved in FLD. L. corymbifera proteins were thus separated by 2D electrophoresis and subjected to western blotting with sera from 7 patients with FLD and 9 healthy exposed controls (HEC). FLD-associated immunoreactive proteins were identified by mass spectrometry based on a protein database specifically created for this study and subsequently produced as recombinant antigens. The ability of recombinant antigens to discriminate patients with FLD from controls was assessed by ELISA performed with sera from FLD patients (n = 41) and controls (n = 43) recruited from five university hospital pneumology departments of France and Switzerland. Forty-one FLD-associated immunoreactive proteins from L. corymbifera were identified. Six of them were produced as recombinant antigens. With a sensitivity and specificity of 81.4 and 77.3% respectively, dihydrolipoyl dehydrogenase was the most effective antigen for discriminating FLD patients from HEC. ELISA performed with the putative proteasome subunit alpha type as an antigen was especially specific (88.6%) and could thus be used for FLD confirmation. The production of recombinant antigens from L. corymbifera represents an additional step towards the development of a standardized ELISA kit for FLD diagnosis.

The thioredoxin system plays a critical role in maintaining the cytoplasm redox state, participating in functions that are important to the cellular viability of fungi. Although functional and structural information on targets in human pathogenic fungi has been scarcely described in the literature, such studies are essential for in silico drug design and biotechnological applications. Therefore, the aims of the present study were to produce recombinant proteins of the thioredoxin system from Candida albicans and evaluate their possible use as prophylactic or alternative therapies against the most important pathogenic fungus associated with nosocomial infections. We focused on biochemical and structural analyses of recombinant thioredoxin reductase from C. albicans with His-tag (CaTrxR-His) for further biotechnology applications. Heterologous CaTrxR-His was efficiently expressed in the soluble fraction of the Escherichia coli lysate. CaTrxR-His was obtained with a high level of purity and presented specific enzymatic activity. Conformational changes of the protein were observed at different pHs and temperatures, with higher thermal stability at pH 8.0. The CaTrxR-His vaccine was shown to effectively induce high levels of CaTrxR-specific immunoglobulin G antibodies in Balb/c mice and reduce the renal fungal burden of experimental disseminated candidiasis in mice. These data may greatly impact future development strategies for vaccine and drug designs against C. albicans infection.

Background: Malassezia commensal yeasts along with multitude of antigens have been found to be associated with various skin disorders including Pityriasis versicolor (PV). Amongst them Mala s1, a 37 kDa protein has been proved to be a major allergen reacting with a large panel of sera. However, there exists no therapeutic alternative to combat such problems in form of plant based natural compounds. The purpose of this study is in the first place, to determine the anti-Malassezia activity of Nyctanthes arbor-tristis L. (NAT) ethanolic leaf extract through turbidimetric growth curves, disruption of plasma membrane and secondly, it aims to present in silico validation of its active constituents over Mala s1a novel allergen., Methods: The antifungal susceptibility 50 % ethanolic extract of NAT was determined by broth microdilution method according to CLSI guidelines. Further MICs and IC50 were determined spectrophotometrically using the software SoftMax® Pro-5 (Molecular Devices, USA). Active constituents mediated disruption of plasma membrane was studied through flowcytometry by permeabilization of fluorescent dye Propidium Iodide (PI). Antioxidant activity of the extract was determined using the DPPH stable radical. Molecular validation of fungal DNA from the extract was observed using PCR amplification. In silico analysis of its active constituents over Mala s1 was performed using HEX software and visualized through Pymol., Results: The anti-Malassezia potential of NAT leaf extracts reflected moderate MIC 1.05 μg/μl against M. globosa, while least effective against M. restricta with MIC 1.47 μg/μl. A linear correlation coefficient R (2) = 0.866 was obtained in case of M. globosa while minimum was observed in M. restricta with R (2) = 0.732. The flow cytometric data reveal ~ 75 % cell death when treated with active constituents β-Sitosterol and Calceolarioside A. The docking confirmations and the interaction energies between Mala s1 and the active constituents (β-Sitosterol and Calceolarioside A) from extracts showed an effective binding which suggests Mala s1 as efficient allergen for site specific targeting., Conclusions: This study revealed that Nyctanthes arbor-tristis L. (NAT) extracts possess high anti-Malassezia potential which is driven mainly by disruption of plasma membrane. Also in silico validation and molecular modeling studies establishes Mala s1 as a novel allergen that could be a potential target in disease treatment. Our results would also provide a foundation for the development of new therapeutic approach using NAT extract as lead compound with high antioxidant property as an added trait for skin care.

The filamentous fungus Lomentospora prolificans is an emerging pathogen causing severe infections mainly among the immunocompromised population. These diseases course with high mortality rates due to great virulence of the fungus, its inherent resistance to available antifungals, and absence of specific diagnostic tools. Despite being widespread in humanized environments, L. prolificans rarely causes infections in immunocompetent individuals likely due to their developed protective immune response. In this study, conidial and hyphal immunomes against healthy human serum IgG were analyzed, identifying immunodominant antigens and establishing their prevalence among the immunocompetent population. Thirteen protein spots from each morph were detected as reactive against at least 70% of serum samples, and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Hence, the most seroprevalent antigens were WD40 repeat 2 protein, malate dehydrogenase, and DHN1, in conidia, and heat shock protein (Hsp) 70, Hsp90, ATP synthase β subunit, and glyceraldehyde-3-phosphate dehydrogenase, in hyphae. More interestingly, the presence of some of these seroprevalent antigens was determined on the cell surface, as Hsp70, enolase, or Hsp90. Thus, we have identified a diverse set of antigenic proteins, both in the entire proteome and cell surface subproteome, which may be used as targets to develop innovative therapeutic or diagnostic tools.

Invasive pulmonary aspergillosis (IPA) is an opportunistic fungal infection in patients undergoing chemotherapy for hematological malignancy, hematopoietic stem cell transplant, or other forms of immunosuppression. In this group, Aspergillus infections account for the majority of deaths due to mold pathogens. Although early detection is associated with improved outcomes, current diagnostic regimens lack sensitivity and specificity. Patients undergoing chemotherapy, stem cell transplantation and lung transplantation were enrolled in a multi-site prospective observational trial. Proven and probable IPA cases and matched controls were subjected to discovery proteomics analyses using a biofluid analysis platform, fractionating plasma into reproducible protein and peptide pools. From 556 spots identified by 2D gel electrophoresis, 66 differentially expressed post-translationally modified plasma proteins were identified in the leukemic subgroup only. This protein group was rich in complement components, acute-phase reactants and coagulation factors. Low molecular weight peptides corresponding to abundant plasma proteins were identified. A candidate marker panel of host response (9 plasma proteins, 4 peptides), fungal polysaccharides (galactomannan), and cell wall components (β-D glucan) were selected by statistical filtering for patients with leukemia as a primary underlying diagnosis. Quantitative measurements were developed to qualify the differential expression of the candidate host response proteins using selective reaction monitoring mass spectrometry assays, and then applied to a separate cohort of 57 patients with leukemia. In this verification cohort, a machine learning ensemble-based algorithm, generalized pathseeker (GPS) produced a greater case classification accuracy than galactomannan (GM) or host proteins alone. In conclusion, Integration of host response proteins with GM improves the diagnostic detection of probable IPA in patients undergoing treatment for hematologic malignancy. Upon further validation, early detection of probable IPA in leukemia treatment will provide opportunities for earlier interventions and interventional clinical trials.

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans, can shield β-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed β-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo. These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: In susceptible individuals, exposure to Aspergillus fumigatus can lead to the development of atopic lung diseases such as allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). Protease allergens including Asp f 5 and Asp f 13 from Aspergillus fumigatus are thought to be important for initiation and progression of allergic asthma., Objective: To assess the importance of secreted protease allergens Asp f 5 (matrix metalloprotease) and Asp f 13 (serine protease) in Aspergillus fumigatus-induced inflammation, airway hyperactivity, atopy and airway wall remodelling in a murine model following chronic exposure to secreted allergens., Methods: BALB/c mice were repeatedly intranasally dosed over the course of 5 weeks with culture filtrate from wild-type (WT), Asp f 5 null (∆5) or Asp f 13 null (∆13) strains of Aspergillus fumigatus. Airway hyper-reactivity was measured by non-invasive whole-body plethysmography, Th2 response and airway inflammation by ELISA and cell counts, whilst airway remodelling was assessed by histological analysis., Results: Parent WT and ∆5 culture filtrates showed high protease activity, whilst protease activity in ∆13 culture filtrate was low. Chronic intranasal exposure to the three different filtrates led to comparable airway hyper-reactivity and Th2 response. However, protease allergen deleted strains, in particular ∆13 culture filtrate, induced significantly less airway inflammation and remodelling compared to WT culture filtrate., Conclusion: Aspergillus fumigatus-secreted allergen proteases, Asp f 5 and Asp f 13, are important for recruitment of inflammatory cells and remodelling of the airways in this murine model. However, deletion of a single allergen protease fails to alleviate airway hyper-reactivity and allergic immune response. Targeting protease activity of Aspergillus fumigatus in conditions such as SAFS or ABPA may have beneficial effects in preventing key aspects of airway pathology., (© 2014 John Wiley & Sons Ltd.)

Nosema bombycis, the first identified microsporidium, is a destructive pathogen of the silkworm Bombyx mori and causes severe worldwide economic losses in sericulture. Major microsporidian structural proteins, such as the spore wall protein (SWP), are known to be involved in host invasion. In this study, the reactivity of the monoclonal antibody 2B10 was tested against an endospore protein of N. bombycis with a molecular weight size at 50-kDa, using Western blotting. The antigen was purified after immunoprecipitation and was further identified as EOB13320 according to MALDI-TOF MS assay. We found that EOB13320 locates to the surface of the different developmental stages of the parasite, mostly the sporoblast stage and the mature spore after immunoelectron microscopy examination. EOB13320 was also widely distributed in the developing endospore, especially at the sporoblast stage. This endospore protein also accumulated in the cytoplasm of both the merogony and sporoblast stages. These results imply that EOB13320 detected by monoclonal antibody 2B10 is expressed throughout the life cycle of the parasite, notably during the stage when the endospore is formed, and that this protein is important for spore-coat formation and parasite maintenance. Our study could be instrumental in the understanding of spore wall formation and will help to gain greater insight into the biology of this parasite.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.

Aspergillus fumigatus is a saprotrophic filamentous fungus and also the most prevalent airborne fungal pathogen of humans. Depending on the host's immune status, the variety of diseases caused by A. fumigatus ranges from allergies in immunocompetent hosts to life-threatening invasive infections in patients with impaired immunity. In contrast to the majority of other Aspergillus species, which are in most cases nonpathogenic, A. fumigatus features an armory of virulence determinants to establish an infection. For example, A. fumigatus is able to evade the human complement system by binding or degrading complement regulators. Furthermore, the fungus interferes with lung epithelial cells, alveolar macrophages, and neutrophil granulocytes to prevent killing by these immune cells. This chapter summarizes the different strategies of A. fumigatus to manipulate the immune response. We also discuss the potential impact of recent advances in immunoproteomics to improve diagnosis and therapy of an A. fumigatus infection.

Invasive aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Diagnosis of this infection frequently employs detection of the circulating galactomannan in the patient serum using enzyme immunoassay (EIA), a highly sensitive and specific system. Although there are many structural studies of the galactomannan of Aspergillus fumigatus, some inconsistencies are present in these results. In this study, to clarify the relationship between the growth conditions and structure of the galactomannans, we cultured A. fumigatus using two distinct yeast/fungal cultivation media, i.e. the yeast extract-peptone-dextrose (YPD) medium and yeast nitrogen base (YNB) medium. Galactomannans prepared from the resulting culture supernatants were structurally characterized by (1)H and (13)C nuclear magnetic resonance, methylation analysis, acetolysis and α-mannosidase degradation. These assays revealed that the galactomannan from the YPD cultivation had short β-1,5-linked galactofuranose (Galf) oligosaccharide chains in both the O- and N-linked carbohydrate moieties, while the galactomannan from the YNB cultivation incorporated long Galf oligosaccharide chains. The galactomannans derived from the two culture conditions significantly differed in reactivity based on the EIA diagnostic system. We also demonstrated the presence of a novel Galf-containing branched oligosaccharide in the O-linked moiety., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii ("Pb01-like") apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43., Methodology: We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients' sera from Southwestern and Midwestern Brazil., Principal Findings: We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species., Conclusion: Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.

Invasive aspergillosis is a life-threatening lung or systemic infection caused by the opportunistic mold Aspergillus fumigatus. The disease affects mainly immunocompromised hosts, and patients with hematological malignances or who have been submitted to stem cell transplantation are at high risk. Despite the current use of Platelia™ Aspergillus as a diagnostic test, the early diagnosis of invasive aspergillosis remains a major challenge in improving the prognosis of the disease. In this study, we used an immunoproteomic approach to identify proteins that could be putative candidates for the early diagnosis of invasive aspergillosis. Antigenic proteins expressed in the first steps of A. fumigatus germination occurring in a human host were revealed using 2-D Western immunoblots with the serum of patients who had previously been classified as probable and proven for invasive aspergillosis. Forty antigenic proteins were identified using mass spectrometry (MS/MS). A BLAST analysis revealed that two of these proteins showed low homology with proteins of either the human host or etiological agents of other invasive fungal infections. To our knowledge, this is the first report describing specific antigenic proteins of A. fumigatus germlings that are recognized by sera of patients with confirmed invasive aspergillosis who were from two separate hospital units.

Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice-enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.

Cryptococcus neoformans is an important human, fungal pathogen that sheds polysaccharide (exo-PS) into host tissues. While shed exo-PS mediates numerous untoward effects (including promoting increased intracranial pressure), little is known about the regulation of this phenomenon. Since downregulation of the Allergen 1 (ALL1) gene is associated with high ICP, we investigated the relationship between ALL1 expression and exo-PS structure using a variety of biophysical techniques. The Δall1 mutants of two serotypes produced a shorter exo-PS with less branching and structural complexity than the parental strains. Consistent with lower branching, these exo-PSs manifested higher intrinsic viscosity than the parental strains. The Δall1 mutant strains manifested differences in epitope expression and significant resistance to phagocytosis. Exo-PS of Δall1 mutant exhibited anti-phagocytic properties. Comparative transcriptome analysis of mutant and parental strain under iron-deprived conditions indicated a role of ALL1 in iron homeostasis, characterized by differential regulation of genes that mediate iron reduction and transport. Together, our results demonstrate a role of ALL1 in regulating conformational aspects of PS structure and iron homeostasis. These findings provide a mechanism to explain how changes in ALL1 expression influence virulence of switch variants and suggest that structural changes and polymer length are epigenetically regulated., (© 2013 John Wiley & Sons Ltd.)

The phytoalexin cyclobrassinin is a plant defense that has additional importance since it inhibits brassinin hydrolase, a phytoalexin detoxifying enzyme produced by the plant pathogen Alternaria brassicicola. Hence, the 1,3-thiazino[6,5-b]indole scaffold of cyclobrassinin has great application as a lead structure to design potential inhibitors of brassinin detoxification. For this reason, it is necessary to determine whether A. brassicicola is able to transform cyclobrassinin. During this work new reactions of 1,3-thiazino[6,5-b]indoles and indoline-2-thiones and their unique [4+2] cycloaddition products were discovered and characterized., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.

Dectin-1 is a pattern recognition receptor that is important for innate immune responses against fungi in humans and mice. Dectin-1 binds to β-glucans in fungal cell walls and triggers phagocytosis, production of reactive oxygen by the NADPH oxidase, and inflammatory cytokine production which all contribute to host immune responses against fungi. Although the autophagy pathway was originally characterized for its role in the formation of double-membrane compartments engulfing cytosolic organelles and debris, recent studies have suggested that components of the autophagy pathway may also participate in traditional phagocytosis. In this study, we show that Dectin-1 signaling in macrophages and bone marrow-derived dendritic cells triggers formation of LC3II, a major component of the autophagy machinery. Further, Dectin-1 directs the recruitment of LC3II to phagosomes, and this requires Syk, activation of reactive oxygen production by the NADPH oxidase, and ATG5. Using LC3-deficient dendritic cells we show that whereas LC3 recruitment to phagosomes is not important for triggering phagocytosis, killing or Dectin-1-mediated inflammatory cytokine production, it facilitates recruitment of MHC class II molecules to phagosomes and promotes presentation of fungal-derived antigens to CD4 T cells.

Background: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall., Methods: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls., Results: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing., Conclusion: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Langerhans cells (LC) are a subset of skin-resident dendritic cells (DC) that reside in the epidermis as immature DC, where they acquire Ag. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli, including epicutaneous sensitization with hapten and skin infection with Candida albicans. Mature LC migrate to the skin-draining LN, where they present Ag to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL-18 on LC. In addition, TLR ligands are present in C. albicans, and hapten sensitization produces endogenous TLR ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88(fl) mice in which LC are insensitive to IL-1 family members and most TLR ligands. LC migration in the steady state, after hapten sensitization and postinfection with C. albicans, was unaffected. Contact hypersensitivity in Langerin-Cre MyD88(fl) mice was similarly unaffected. Interestingly, in response to C. albicans infection, these mice displayed reduced proliferation of Ag-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of costimulatory molecules was intact on LC, but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration, but is required for the full activation and function of LC in the setting of fungal infection.

CD4(+) T cells are the key players of vaccine resistance to fungi. The generation of effective T cell-based vaccines requires an understanding of how to induce and maintain CD4(+) T cells and memory. The kinetics of fungal antigen (Ag)-specific CD4(+) T cell memory development has not been studied due to the lack of any known protective epitopes and clonally restricted T cell subsets with complementary T cell receptors (TCRs). Here, we investigated the expansion and function of CD4(+) T cell memory after vaccination with transgenic (Tg) Blastomyces dermatitidis yeasts that display a model Ag, Eα-mCherry (Eα-mCh). We report that Tg yeast led to Eα display on Ag-presenting cells and induced robust activation, proliferation, and expansion of adoptively transferred TEa cells in an Ag-specific manner. Despite robust priming by Eα-mCh yeast, antifungal TEa cells recruited and produced cytokines weakly during a recall response to the lung. The addition of exogenous Eα-red fluorescent protein (RFP) to the Eα-mCh yeast boosted the number of cytokine-producing TEa cells that migrated to the lung. Thus, model epitope expression on yeast enables the interrogation of Ag presentation to CD4(+) T cells and primes Ag-specific T cell activation, proliferation, and expansion. However, the limited availability of model Ag expressed by Tg fungi during T cell priming blunts the downstream generation of effector and memory T cells.

Background: Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13., Methodology/principal Findings: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A(148)-E(166)) and S22 (A(243)-K(274)) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T(261)-K(274)), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity., Conclusions/significance: Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.

Because of the potential protective role of leukotrienes (LTs) in histoplasmosis and the therapeutic and prophylactic effects of cell-free antigens from Histoplasmacapsulatum (CFAgs), the aim of this study was to develop and characterise biodegradable LTB(4)/CFAgs-loaded microspheres (MS) that could promote cellular activation for future immunisation purposes. LTB(4)/CFAgs-loaded MS that were developed through a double emulsion/extraction process were characterised according to their size, zeta potential, morphology, entrapment efficiency and in vitro release kinetics. We evaluated the uptake of LTB(4)/CFAgs-loaded MS by bone marrow derived-macrophages (BMDM). The TNF-α and chemokines, and nitrite production, in the supernatant of BMDM cultures were analysed by enzyme-linked immunosorbent assay (ELISA) and Griess reaction, respectively. We found an instantaneous release of CFAgs and a prolonged release of LTB(4) from the poly-(d,l-lactide-co-glycolide) (PLGA) MS. The microencapsulation process did not alter the zeta potential nor the spherical morphology of the MS. The appropriate size of the LTB(4)/CFAgs-loaded MS (smaller than 10μm) enabled the efficient uptake by BMDM and also induced TNF-α, CXCL1/KC, CCL2/MCP-1, CCL5/RANTES and nitrite oxide release by these cells. In conclusion, the biodegradable LTB(4)/CFAgs-loaded MS were able to efficiently activate murine BMDM and thereby have the potential to be used in an effective vaccine against H. capsulatum infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

p27 is an antigenic protein produced by Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis (PCM). Despite its unknown function, it has been suggested as a putative virulence factor, proposed as a suitable target for the design of diagnostic tools and vaccines, and considered as an enhancer in antifungal treatment of PCM. We evaluated sequence polymorphisms of PbP27 gene sequence among isolates, finding some polymorphisms associated with the isolates' phylogenetic origin. In order to determine if there was a differential expression pattern between morphological states and among isolates, we also evaluated PbP27 expression, at transcriptional and translational levels, in mycelia and yeast cultures in 14 isolates belonging to the P. brasiliensis species complex (S1, PS2, PS3, and "Pb01-like", proposed to be named Paracoccidioides lutzii) by two techniques, real time RT-PCR (RT-qPCR) and protein dot blot. For the latter, four protein extracts from different cell localizations (SDS or β-mercaptoethanol, cytoplasmic and extracellular proteins) were analyzed for each isolate. p27 was present in the four extracts evaluated, mainly in the SDS extract, corresponding to an extract containing proteins loosely attached to the cell wall. This information correlates with immunohistochemical analysis, where positive staining of the yeasts' cell wall was observed. We found that p27 was present in all isolates, mainly in the yeast form. This pattern was corroborated by RT-qPCR results, with higher expression levels found in the yeast form for most of the isolates. The results provide new insights into the expression patterns of this protein, and further characterize it in view of potential uses as a diagnostic and/or therapeutic tool., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The glycoprotein gp43 is an immunodominant antigen secreted by Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. The present study evaluated whether gp43 can interact with toll-like (TLR2, TLR4) and mannose (MR) receptors on the surface of human monocytes, and how that affects their expression and cytokine production. Monocytes were incubated with or without monoclonal antibodies anti-TLR2, anti-TLR4, or anti-MR, individually or in combination, prior to the addition of gp43. The gp43 binding to monocyte surface, as well as expression of TLR2, TLR4, and MRs were analyzed by flow cytometry, while production of TNF-α and IL-10 was monitored by ELISA. The results suggested that gp43 binds to TLR2, TLR4, and MR receptors, with TLR2 and MR having the strongest effect. All three receptors influenced the production of IL-10, while TNF-α production was associated with expression of TLR4 and MR. The modulatory effect of gp43 was demonstrated by high levels of TLR4 expression associated with increased production of TNF-α after 4 h of culture. Alternatively, high levels of TLR2 expression, and elevated production of IL-10, were detected after 18 h. We showed that interaction between gp43 and monocytes may affect the innate immune response by modulating the expression of the pattern recognition receptors TLR2, TLR4 and MR, as well as production of pro- and anti-inflammatory cytokines.