Synthetic Glycans Reveal Determinants of Antibody Functional Efficacy against a Fungal Pathogen.

Antibodies play a vital role in the immune response to infectious diseases and can be administered passively to protect patients. In the case of Cryptococcus neoformans, a WHO critical priority fungal pathogen, infection results in antibodies targeting capsular glucuronoxylomannan (GXM). These antibodies yield protective, non-protective, and disease-enhancing outcomes when administered passively. However, it was unknown how these distinct antibodies recognized their antigens at the molecular level, leading to the hypothesis that they may target different GXM epitopes. To test this hypothesis, we constructed a microarray containing 26 glycans representative of those found in highly virulent cryptococcal strains and utilized it to study 16 well-characterized monoclonal antibodies. Notably, we found that protective and non-protective antibodies shared conserved reactivity to the M2 motif of GXM, irrespective of the strain used in infection or GXM-isolated to produce a conjugate vaccine. Here, only two antibodies, 12A1 and 18B7, exhibited diverse trivalent GXM motif reactivity. IgG antibodies associated with protective responses showed cross-reactivity to at least two GXM motifs. This molecular understanding of antibody binding epitopes was used to map the antigenic diversity of two Cryptococcus neoformans strains, which revealed the exceptional complexity of fungal capsular polysaccharides. A multi-GXM motif vaccine holds the potential to effectively address this antigenic diversity. Collectively, these findings underscore the context-dependent nature of antibody function and challenge the classification of anti-GXM epitopes as either "protective" or "non-protective".

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

Fecal Bacteria Implicated in Biofilm Production Are Enriched and Associate to Gastrointestinal Symptoms in Patients With APECED - A Pilot Study.

Backgrounds and Aims: APECED is a rare autoimmune disease caused by mutations in the Autoimmune Regulator gene. A significant proportion of patients also have gastrointestinal symptoms, including malabsorption, chronic diarrhea, and obstipation. The pathological background of the gastrointestinal symptoms remains incompletely understood and involves multiple factors, with autoimmunity being the most common underlying cause. Patients with APECED have increased immune responses against gut commensals. Our objective was to evaluate whether the intestinal microbiota composition, predicted functions or fungal abundance differ between Finnish patients with APECED and healthy controls, and whether these associate to the patients' clinical phenotype and gastrointestinal symptoms. Methods: DNA was isolated from fecal samples from 15 patients with APECED (median age 46.4 years) together with 15 samples from body mass index matched healthy controls. DNA samples were subjected to analysis of the gut microbiota using 16S rRNA gene amplicon sequencing, imputed metagenomics using the PICRUSt2 algorithm, and quantitative PCR for fungi. Extensive correlations of the microbiota with patient characteristics were determined. Results: Analysis of gut microbiota indicated that both alpha- and beta-diversity were altered in patients with APECED compared to healthy controls. The fraction of Faecalibacterium was reduced in patients with APECED while that of Atopobium spp. and several gram-negative genera previously implicated in biofilm formation, e.g. Veillonella, Prevotella, Megasphaera and Heamophilus, were increased in parallel to lipopolysaccharide (LPS) synthesis in imputed metagenomics. The differences in gut microbiota were linked to patient characteristics, especially the presence of anti-Saccharomyces cerevisiae antibodies (ASCA) and severity of gastrointestinal symptoms. Conclusions: Gut microbiota of patients with APECED is altered and enriched with predominantly gram-negative bacterial taxa that may promote biofilm formation and lead to increased exposure to LPS in the patients. The most pronounced alterations in the microbiota were associated with more severe gastrointestinal symptoms.

Rheological properties of cryptococcal polysaccharide change with fiber size, antibody binding and temperature.

Aim:Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.

Development of Mold Antigen-Specific Enzyme-Linked Immunosorbent Assays (ELISA) to Quantify Airborne Antigen Exposure.

Common methods to quantify molds in the environment are based on the detection of viable and nonviable fungal components using cultivation technique or assessment by microscopy. These methods are time consuming and laborious and require a high expertise and especially in airborne exposure studies they showed poor reproducibility. Therefore alternative techniques based on molecular or immunological tools attract wide interest. The development of specific ELISAs based on polyclonal antibodies to detect mold antigens in airborne samples starting with the extraction of the antigen material up to evaluation of the sandwich ELISA is summarized in this chapter.

Allergic bronchopulmonary aspergillosis presumably unmasked by PD-1 inhibition.

Programmed cell death-1 (PD-1) inhibitors stimulate immune recognition of tumour cells in cancer patients, but have significant autoimmune side effects including pneumonitis. We report the case of a patient with asthma and mild eosinophilia who developed unusual pulmonary side effect of bronchiectasis, severe eosinophilia (absolute eosinophil count: 3200 c/mm3) and elevated IgE levels (7050 IU/mL; normal: <164 IU/mL) 4 months into therapy with the PD-1 inhibitor pembrolizumab. Aspergillus fumigatus IgG was elevated at 15.60 U/mL (normal: <12.01 U/mL). He responded to therapy with corticosteroids and voriconazole and was able to resume pembrolizumab thereafter with good clinical response.

A novel monoclonal antibody against enolase antigen of Aspergillus fumigatus protects experimental aspergillosis in mice.

Aspergillus fumigatus is one of the most common opportunistic fungal pathogens responsible for a variety of diseases in human, from allergic bronchopulmonary aspergillosis to chronic pulmonary aspergillosis, mostly in immunocompromized patients. In this study, one monoclonal antibody MAb R-5 (IgM) raised against enolase cell surface protein of A. fumigatus exhibited significant inhibition of spore germination in A. fumigatus (88.3%), Aspergillus flavus (57.4%) and Aspergillus niger (30.6%). The MAb R-5 also showed in vitro fungicidal activity against these species as follows: A. fumigatus (24.1%), A. flavus (13.3%) and A. niger (8.8%). These findings were supported by the indirect immunofluorescence microscopy, where the antibody showed binding with germinated spores and hyphae of A. fumigatus as well as A. flavus and A. niger.In vivo protective effect of MAb R-5 was evaluated in BALB/c mice challenged intravenously with A. fumigatus spores, where a significant reduction in CFU (85.9%) was observed in kidney tissue. The mean survival time of mice treated with MAb R-5 (18.5 days) was also enhanced compared to control (6.5 days). These results indicate that MAb R-5 could be valuable in diagnosis as well as in the treatment of broad range of Aspergillus infections.

Mold allergy: is it real and what do we do about it?

INTRODUCTION: fungi produce substances that contain pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps) which bind to pattern recognition receptors, stimulating innate immune responses in humans. they also produce allergens that induce production of specific ige. Areas covered: In this review we cover both innate and adaptive immune responses to fungi. Some fungal products can activate both innate and adaptive responses and in doing so, cause an intense and complex health effects. Methods of testing for fungal allergy and evidence for clinical treatment including environmental control are also discussed. In addition, we describe controversial issues including the role of Stachybotrys and mycotoxins in adverse health effects. Expert commentary: Concerns about long-term exposure to fungi have led some patients, attorneys and fungus advocates to promote fears about a condition that has been termed toxic mold syndrome. This syndrome is associated with vague symptoms and is believed to be due to exposure to mycotoxins, though this connection has not been proven. Ultimately, more precise methods are needed to measure both fungal exposure and the resulting health effects. Once that such methods become available, much of the speculation will be replaced by knowledge.

Immunolocalization of Proteins in Fission Yeast by Electron Microscopy.

Electron microscopy (EM) immunolocalization of antigens in fission yeast can be accomplished with cells processed by rapid freezing and freeze-substitution followed by embedding in acrylic or methacrylate resins. Microtome sections of embedded cells are collected onto EM grids. Primary antibodies to the antigen of interest, followed by secondary antibodies conjugated to colloidal gold, are allowed to bind to antigens at the surface of these plastic sections. This type of postembed labeling provides information on antigen localization to a resolution of 10-20 nm, depending on the size of the metal particle used, the form of the antibody (Fab vs. complete IgG or IgM), and whether direct or indirect labeling is used. The method has the potential to map macromolecules in three dimensions in a relatively large volume when thin (30-60-nm) serial sections are labeled, imaged, aligned, and modeled to create a representative volume. The biggest challenge of this technique is the necessary compromise between the preservation of cellular ultrastructure and the preservation of antigen reactivity. The protocols described here show how to immunolabel samples for EM and include suggestions for overcoming challenges related to antigen preservation.

Assessment of Fecal ASCA Measurement as a Biomarker of Crohn Disease in Pediatric Patients.

OBJECTIVES: A simple and reliable biomarker for Crohn disease (CD) would be a valuable clinical tool. We hypothesized that anti-Saccharomyces cerevisiae antibody (ASCA) may be present in the stool of patients with CD. Accordingly, we measured ASCA in the stool and serum of children and adolescents with known or suspected inflammatory bowel disease (IBD). METHODS: We included 114 patients 19 years or younger (73 boys) with IBD, including 83 patients with CD and 31 subjects without CD (28 with ulcerative colitis, and 3 patients with suspected IBD but without evidence of chronic inflammation at the time of their endoscopy and colonoscopy). Fecal and serum samples were analyzed using semiquantitative ASCA enzyme-linked immunoassays. RESULTS: Median ASCA levels were significantly elevated in the stool (P = 0.04) and serum (P = 0.0008) of patients with CD, when compared to levels observed in patients without CD. Fecal ASCA levels were similarly more elevated in patients with active CD, relative to levels observed in patients with active ulcerative colitis and acute colitis (P = 0.004). Among patients with CD, fecal and serum ASCA levels were higher (P = 0.01 and 0.01, respectively) in patients with more recently diagnosed disease. CONCLUSIONS: Fecal ASCA levels are higher in patients with active and newly diagnosed disease. Data from the present study suggest that measurement of fecal ASCA levels could represent a novel noninvasive biomarker for use in evaluating patients with suspected or known IBD. Further studies are necessary to better define the value of fecal ASCA measurements in identifying CD and response to therapy in children and young adults.

Polyclonal anti-Candida antibody improves phagocytosis and overall outcome in zebrafish model of disseminated candidiasis.

Fungal infections are a major cause of animal and plant morbidity and mortality worldwide. Effective biological therapeutics could complement current antifungal drugs, but understanding of their in vivo mechanisms has been hampered by technical barriers to intravital imaging of host-pathogen interactions. Here we characterize the fungal infection of zebrafish as a model to understand the mechanism-of-action for biological antifungal therapeutics through intravital imaging of these transparent animals. We find that non-specific human IgG enhances phagocytosis by zebrafish phagocytes in vivo. Polyclonal anti-Candida antibodies enhance containment of fungi in vivo and promote survival. Analysis suggests that early phagocytic containment is a strong prognostic indicator for overall survival. Although polyclonal anti-Candida antibodies protect against disease, this is not necessarily the case for individual monoclonal anti-Candida antibodies. Thus, the zebrafish appears to provide a useful model host for testing if a biological therapeutic promotes phagocytosis in vivo and enhances protection against candidemia.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

The glycolytic enzyme enolase represents a plasminogen-binding protein on the surface of a wide variety of medically important fungal species.

Allergies are an increasing issue in human health and can, eventually, cause severe anaphylactic shock. Aspergillus fumigatus and Candida albicans are leading causes of life-threatening invasive fungal infections in immunocompromised patients, but can also cause severe allergic responses in otherwise healthy individuals. The glycolytic enzyme enolase is known as a major allergen despite its function in intracellular metabolism. Therefore, its presentation on surfaces of different fungal species was investigated by using antibodies raised against recombinant enolases from A. fumigatus and C. albicans. Examination of antibody specificity revealed cross-reactivity to cell-free extracts from Aspergillus terreus, Aspergillus flavus, Aspergillus nidulans and Candida glabrata, but not against any of the three human enolases. Antibody specificity was further confirmed by hybridization with other recombinant fungal enolases, where the antibodies recognized different subsets of fungal enolases. When surface presentation of enolase was tested on intact fungal cells, a positive staining was obtained with those antibodies that also recognized the enzyme from the respective cell-free extract. This implies a general surface presentation of this glycolytic enzyme among fungal species and provides hints for its predominant recognition as an allergen. Additionally, A. fumigatus and C. albicans enolase bound to human plasminogen, which remained accessible for the plasminogen activator uPA. This implies a potential role of enolase in the invasion and dissemination process during fungal infections.

A monoclonal antibody that tracks endospore formation in the microsporidium Nosema bombycis.

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.

Inhibition of Yeast Growth by Broadly Cross-Reactive Antisera Elicited by Heterologous Mannan-Protein Conjugate.

A new approach to obtain broadly cross-reactive antisera against important yeast pathogens by intensive hyperimmunization with polysaccharide-protein conjugates is described here. Surface mannan of Candida albicans and capsular galactoglucoxylomannan of Cryptococcus laurentii were isolated and chemically linked to human serum albumin. Antisera elicited by a 7-week vigorous immunization of rabbits with the conjugates showed effective cross-reactive growth inhibition of different representatives of Candida spp. as well as Cryptococcus spp. IgG antibodies are evidenced as the effective component of the antisera.

Changes in atmospheric CO2 influence the allergenicity of Aspergillus fumigatus.

Increased susceptibility to allergies has been documented in the Western world in recent decades. However, a comprehensive understanding of its causes is not yet available. It is therefore essential to understand trends and mechanisms of allergy-inducing agents, such as fungal conidia. In this study, we investigated the hypothesis that environmental conditions linked to global atmospheric changes can affect the allergenicity of Aspergillus fumigatus, a common allergenic fungal species in indoor and outdoor environments and in airborne particulate matter. We show that fungi grown under present-day CO2 levels (392 ppm) exhibit 8.5 and 3.5 fold higher allergenicity compared to fungi grown at preindustrial (280 ppm) and double (560 ppm) CO2 levels, respectively. A corresponding trend is observed in the expression of genes encoding for known allergenic proteins and in the major allergen Asp f1 concentrations, possibly due to physiological changes such as respiration rates and the nitrogen content of the fungus, influenced by the CO2 concentrations. Increased carbon and nitrogen levels in the growth medium also lead to a significant increase in the allergenicity. We propose that climatic changes such as increasing atmospheric CO2 levels and changes in the fungal growth medium may impact the ability of allergenic fungi such as A. fumigatus to induce allergies.

Antibody binding to Cryptococcus neoformans impairs budding by altering capsular mechanical properties.

Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions ("quellung" effects). A problem in studying Ab-capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab-capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system.

Actividad antifúngica de la altenusina aislada del hongo endofítico Alternaria sp. frente al hongo patógeno Paracoccidioides brasiliensis / Antifungal activity of altenusin isolated from the endophytic fungus Alternaria sp. against the pathogenic fungus Paracoccidioides brasiliensis

Antecedentes. La altenusina es un derivado bifenilo aislado de diferentes especies de hongos, que presenta una diversidad de actividades biológicas. Objetivos. Describimos la actividad antifúngica de la altenusina aislada del hongo endofítico Alternaria sp. frente a aislamientos clínicos de Paracoccidioides brasiliensis, y su acción sobre las paredes celulares de P. brasiliensis y la levadura no patógena Schizosaccharomyces pombe. Métodos. Se valoró la actividad antifúngica de la altenusina in vitro usando un método de microdilución en caldo frente a 11 cepas de P. brasiliensis y una cepa de S. pombe. Los efectos de la altenusina sobre la pared celular se estimaron utilizando un análisis de protección con sorbitol. Resultados. La altenusina presentó una potente actividad frente a P. brasiliensis con valores de concentración inhibitoria mínima (CIM) que variaron entre 1,9 y 31,2μg/ml, y de 62,5μg/ml para S. pombe. Los resultados del presente estudio demostraron que los valores CIM de la altenusina aumentaron para Pb18 de P. brasiliensis y para S. pombe cuando el medio se suplementó con sorbitol. Además, las células de S. pombe tratadas con altenusina adoptaron una forma más redondeada que las no tratadas. Conclusiones. Con la concentración examinada, la altenusina demostró actividad frente a las cepas clínicas de P. brasiliensis, y es probable que este preparado afecte a las paredes de las células micóticas. Estos hallazgos sugieren que la altenusina podría actuar a través de la inhibición de la síntesis o ensamblado de la pared celular en P. brasiliensis y S. pombe y podría considerarse la molécula inicial para el diseño de nuevos antimicóticos(AU)

Background. Altenusin is a biphenyl derivative isolated from different species of fungi, which presents several biological activities. Aims. We report the antifungal activity of the altenusin isolated from the endophytic fungus Alternaria sp., against clinical isolates of Paracoccidioides brasiliensis, and its action on cell walls of P. brasiliensis and the nonpathogenic yeast Schizosaccharomyces pombe. Methods. In vitro antifungal activity of altenusin was evaluated using the broth microdilution method against 11 strains of P. brasiliensis and one strain of S. pombe. The effects of the altenusin on the cell wall were estimated using the sorbitol protection assay. Results. The altenusin presented strong activity against P. brasiliensis with MIC values ranging between 1.9 and 31.2μg/ml, and 62.5μg/ml for S. pombe. Our results demonstrated that the MIC values for altenusin were increased for P. brasiliensis Pb18 and for S. pombe when the medium was supplemented with sorbitol. Additionally, S. pombe cells treated with altenusin were more rounded in shape than untreated cells. Conclusions. Altenusin showed activity against clinical strains of P. brasiliensis at the concentration tested, and this compound probably affects fungal cell walls. These findings suggest that altenusin could act through the inhibition of cell wall synthesis or assembly in P. brasiliensis and S. pombe, and could be considered as a lead compound for the design of new antifungals(AU)