Bioengineering a plant NLR immune receptor with a robust binding interface toward a conserved fungal pathogen effector.

Bioengineering of plant immune receptors has emerged as a key strategy for generating novel disease resistance traits to counteract the expanding threat of plant pathogens to global food security. However, current approaches are limited by rapid evolution of plant pathogens in the field and may lack durability when deployed. Here, we show that the rice nucleotide-binding, leucine-rich repeat (NLR) immune receptor Pik-1 can be engineered to respond to a conserved family of effectors from the multihost blast fungus pathogen Magnaporthe oryzae. We switched the effector binding and response profile of the Pik NLR from its cognate rice blast effector AVR-Pik to the host-determining factor pathogenicity toward weeping lovegrass 2 (Pwl2) by installing a putative host target, OsHIPP43, in place of the native integrated heavy metal-associated domain (generating Pikm-1OsHIPP43). This chimeric receptor also responded to other PWL alleles from diverse blast isolates. The crystal structure of the Pwl2/OsHIPP43 complex revealed a multifaceted, robust interface that cannot be easily disrupted by mutagenesis, and may therefore provide durable, broad resistance to blast isolates carrying PWL effectors in the field. Our findings highlight how the host targets of pathogen effectors can be used to bioengineer recognition specificities that have more robust properties compared to naturally evolved disease resistance genes.

Hyphal Als proteins act as CR3 ligands to promote immune responses against Candida albicans.

Patients with decreased levels of CD18 (ß2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMß2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the ß-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the "outside-in signaling" for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1ß release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.

[Diagnosis of allergen components in fungi and microbial communities: patient sensitization characteristics and main sensitizing proteins].

The human body, as a highly complex ecosystem, harbors diverse microbial communities, with major factors triggering allergic reactions encompassing the skin microbiome and fungi. The global diversity of fungi is estimated to range from approximately 600 000 to 1 million species, and theoretically, IgE-mediated sensitization may occur to any fungal species. As of now, the World Health Organization/IUIS official database records 113 fungal allergens originating from 30 different fungi species, covering 42 allergen families. Regarding the skin microbiome, 14 distinct Malassezia allergens have been identified, all derived from three different Malassezia fungi species--M. furfur, M. sympodialis, and M. globosa. The conditions of patients with these allergies are exceptionally complex. This article extensively discusses the latest research advancements and clinical applications related to skin microbiome and fungal allergies from the European Academy of Allergy and Clinical Immunology (EAACI) publication, "Molecular Allergology User's Guide 2.0". Additionally, it compiles information on the sources of fungal allergens, characteristics of allergen component protein families, clinical relevance, and management strategies, both domestically and internationally. The aim is to enhance the profound understanding of allergen components among relevant professionals. Through the application of advanced allergen component diagnostic techniques, the goal is to achieve precise diagnosis and treatment of fungal allergy patients and explore the mechanisms underlying fungal sensitization and pathogenesis, laying the foundation for studying the fungal allergen protein sensitization spectrum in the Chinese population.

Design of a cryptococcus neoformans vaccine by subtractive proteomics combined with immunoinformatics.

The emergence of Cryptococcus neoformans has posed an undeniable burden to many regions worldwide, with its strains mainly entering the lungs through the respiratory tract and spreading throughout the body. Limitations of drug regimens, such as high costs and limited options, have directed our attention toward the promising field of vaccine development. In this study, the subtractive proteomics approach was employed to select target proteins from databases that can accurately cover serotypes A and D of the Cryptococcus neoformans. Further, two multi-epitope vaccines consisting of T and B cell epitopes were demonstrated that they have good structural stability and could bind with immune receptor to induce desired immune responses in silico. After further evaluation, these vaccines show the potential for large-scale production and applicability to the majority of the population of the world. In summary, these two vaccines have been theoretically proven to combat Cryptococcus neoformans infections, awaiting further experimental validation of their actual protective effects.

A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Identification of the Most Immunoreactive Antigens of Candida auris to IgGs from Systemic Infections in Mice.

The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.

[In silico analysis of molecular mimicry between human aquaporin 3, Aspergillus fumigatus aquaporin and aquaporins from allergic sources]. / Análisis en silico del mimetismo molecular entre acuaporina 3 humana y Aspergillus fumigatus: implicaciones para el potencial de reactividad cruzada.

OBJECTIVE: Conduct an in-silico assessment of potential molecular mimicry between human aquaporins, A. fumigatus, and diverse allergenic sources. METHODS: Amino acid sequences of human AQP3 and A. fumigatus aquaporin were compared through multiple alignments with 25 aquaporins from diverse allergenic sources. Phylogenetic analysis and homology-based modeling were executed, and the ElliPro server predicted conserved antigenic regions on 3D structures. RESULTS: Global identity among studied aquaporins was 32.6%, with a specific conserved local region at 71.4%. Five monophyletic clades (A-E) were formed, and Group B displayed the highest identity (95%), including 6 mammalian aquaporins, notably AQP3. A. fumigatus aquaporin exhibited the highest identity with Malassezia sympodialis (35%). Three linear and three discontinuous epitopes were identified in both human and A. fumigatus aquaporins. The Root Mean Square Deviation (RMSD) from overlapping aquaporin structures was 1.006. CONCLUSION: Identification of potential linear and conformational epitopes on human AQP3 suggests likely molecular mimicry with A. fumigatus aquaporins. High identity in a specific antigenic region indicates potential autoreactivity and a probable antigenic site involved in cross-reactivity. Validation through in vitro and in vivo studies is essential for further understanding and confirmation.

OBJETIVO: Realizar una evaluación in silico del posible mimetismo molecular entre las acuaporinas humanas, A. fumigatus y diversas fuentes alergénicas. MÉTODOS: Se compararon secuencias de aminoácidos de AQP3 humana y acuaporina de A. fumigatus mediante alineamientos múltiples con 25 acuaporinas de diversas fuentes alergénicas. Se ejecutaron análisis filogenéticos y modelos basados en homología, y el servidor ElliPro predijo regiones antigénicas preservadas en estructuras 3D. RESULTADOS: La identidad global entre las acuaporinas estudiadas fue del 32.6%, con una región local específica preservada en el 71.4%. Se formaron cinco clados monofiléticos (A-E), y el grupo B mostró la identidad más alta (95%), incluidas 6 acuaporinas de mamíferos, en particular AQP3. A. fumigatus aquaporin exhibió la mayor identidad con Malassezia sympodialis (35%). Se identificaron tres epítopos lineales y tres discontinuos en acuaporinas tanto humanas como de A. fumigatus. La desviación cuadrática media (RMSD) de las estructuras de acuaporinas superpuestas fue de 1,006. CONCLUSIÓN: La identificación de posibles epítopos lineales y conformacionales en AQP3 humano sugiere un probable mimetismo molecular con acuaporinas de A. fumigatus. La identidad alta en una región antigénica específica indica autorreactividad potencial y un sitio antigénico probable implicado en la reactividad cruzada. La validación mediante estudios in vitro e in vivo es desicivo para una mayor comprensión y confirmación.

In silico evaluation of the potential allergenicity of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

The world's hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

Vaccination with structurally adapted fungal protein fibrils induces immunity to Parkinson's disease.

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

Identification of allergens from Aspergillus fumigatus-Potential association with lung damage in asthma.

BACKGROUND: Component-resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2-dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained. METHODS: We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti-human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract. RESULTS: Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus-sensitized people with asthma, 23 non-fungal-sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose-bisphosphate aldolase class II (FBA2) (33%), NAD-dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens. CONCLUSION: In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.

Interactions of Candida tropicalis pH-related antigen 1 with complement proteins C3, C3b, factor-H, C4BP and complement evasion.

Candida, as a part of the human microbiota, can cause opportunistic infections that are either localised or systemic candidiasis. Emerging resistance to the standard antifungal drugs is associated with increased mortality rate due to invasive Candida infections, particularly in immunocompromised patients. While there are several species of Candida, an increasing number of Candida tropicalis isolates have been recently reported from patients with invasive candidiasis or inflammatory bowel diseases. In order to establish infections, C. tropicalis has to adopt several strategies to escape the host immune attack. Understanding the immune evasion strategies is of great importance as these can be exploited as novel therapeutic targets. C. albicans pH-related antigen 1 (CaPra1), a surface bound and secretory protein, has been found to interact strongly with the immune system and help in complement evasion. However, the role of C. tropicalis Pra1 (CtPra1) and its interaction with the complement is not studied yet. Thus, we characterised how pH-related antigen 1 of C. tropicalis (CtPra1) interacts with some of the key complement proteins of the innate immune system. CtPra1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. Recombinant CtPra1, was found to bind human C3 and C3b, central molecules of the complement pathways that are important components of the innate immune system. It was also found to bind human complement regulatory proteins factor-H and C4b-binding protein (C4BP). CtPra1-factor-H and CtPra1-C4BP interactions were found to be ionic in nature as the binding intensity affected by high sodium chloride concentrations. CtPra1 inhibited functional complement activation with different effects on classical (∼20 %), lectin (∼25 %) and alternative (∼30 %) pathways. qPCR experiments using C. tropicalis clinical isolates (oral, blood and peritoneal fluid) revealed relatively higher levels of expression of CtPra1 gene when compared to the reference strain. Native CtPra1 was found to be expressed both as membrane-bound and secretory forms in the clinical isolates. Thus, C. tropicalis appears to be a master of immune evasion by using Pra1 protein. Further investigation using in-vivo models will help ascertain if these proteins can be novel therapeutic targets.

BACH2 in TRegs Limits the Number of Adipose Tissue Regulatory T Cells and Restrains Type 2 Immunity to Fungal Allergens.

FoxP3+ regulatory T cells (Tregs) are essential for self-tolerance and moderating tissue-damaging inflammation. Tregs that develop and mature in the thymus are classified as central Tregs or effector Tregs based on whether Tregs predominately inhabit secondary lymphoid organs (central Tregs) or tissues (effector Tregs). By generating mice that are conditionally deficient for Bach2 in peripheral Tregs, we have examined the role of Bach2 in regulating Treg homeostasis and effector functions. Unlike global and T cell-specific Bach2-deficient mice, Treg-specific Bach2 ablation did not result in unprovoked TH2 inflammation in the lungs. However, Bach2 deficiency in Tregs led to augmented expressions of IRF4, BATF, and GATA3 and a significant increase in the accumulation of ST2 (IL-33R)+ve effector Tregs in the spleen and visceral adipose tissue (VAT) but not in the lungs. Enhanced Bach2-deficient Treg numbers in VAT was not linked to hyperresponsiveness to exogenous IL-33 in vivo. Most strikingly, Treg-specific Bach2 deficiency resulted in enhanced fungal protease-induced Type 2 allergic inflammation in the lungs, with no detectable effects on Type 1 responses to systemic or respiratory viral infections. In summary, we ascribe vital roles for Bach2 in peripheral Tregs: as a transcriptional checkpoint to limit precocious differentiation into effector Tregs in lymphoid tissues and as a regulator of the functional program that restrains Type 2 but not Type 1 inflammation in lungs. Results presented in this manuscript implicate dysregulated Tregs in the pathogenesis of airway hypersensitivities, asthma, and other allergic disorders.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Development and validation of monoclonal antibodies specific for Candida albicans Als2, Als9-1, and Als9-2.

Fungal agglutinin-like sequence (Als) cell-surface glycoproteins, best characterized in Candida albicans, mediate adhesive and aggregative interactions with host cells, other microbes, and abiotic surfaces. Monoclonal antibodies (MAbs) specific for each C. albicans Als protein are valuable reagents for gaining insight into Als protein localization and function. This manuscript describes development and validation of MAbs specific for C. albicans Als2, as well as for C. albicans Als9-1 and Als9-2, two protein variants produced from the ALS9 locus. Native C. albicans ALS9 expression levels were not sufficiently high to produce detectable Als9 protein on the wild-type cell surface so MAb validation required production of overexpression strains, each featuring one of the two ALS9 alleles. An anti-Als2 MAb was raised against an N-glycosylated form of the protein immunogen, as well as an Endoglycosidase H-treated immunogen. The MAb raised against the N-glycosylated immunogen proved superior and immunolabeled C. albicans yeast cells and germ tubes, and the surface of Candida dubliniensis and Candida tropicalis yeasts. Als2 was visible on C. albicans yeast cells recovered from a murine model of oral candidiasis, demonstrating Als2 production both in vivo and in vitro. These new MAbs add to the collection of anti-Als MAbs that are powerful tools to better understand the role of Als proteins in C. albicans biology and pathogenesis.

Alt a 1 Promotes Allergic Asthma In Vivo Through TLR4-Alveolar Macrophages.

The mold Alternaria alternata is one of the main sources of asthma exacerbation, being its major allergen, Alt a 1, indispensable for its development. The main objective of this work was to answer two main questions: 1) can Alt a 1 by itself (without any other context) induce an asthmatic profile in vivo?; and 2) Which molecular mechanisms take place during this phenomenon? To answer both questions, we have developed a mouse model of allergic asthma using only Alt a 1 for mice sensitization. We also made use of in-vitro cellular models and computational studies to support some aspects of our hypothesis. Our results showed that Alt a 1 can induce an asthmatic phenotype, promoting tissue remodeling and infiltration of CD45+ cells, especially eosinophils and macrophages (Siglec F+ and F4/80+). Also, we have found that Alt a 1 sensitization is mediated by the TLR4-macrophage axis.

Epitope-Based Immunoinformatic Approach on Heat Shock 70 kDa Protein Complex of Cryptococcus neoformans var. grubii.

INTRODUCTION: Cryptococcosis is a ubiquitous opportunistic fungal disease caused by Cryptococcus neoformans var. grubii. It has high global morbidity and mortality among HIV patients and non-HIV carriers with 99% and 95%, respectively. Furthermore, the increasing prevalence of undesired toxicity profile of antifungal, multidrug-resistant organisms and the scarcity of FDA-authorized vaccines were the hallmark in the present days. This study was undertaken to design a reliable epitope-based peptide vaccine through targeting highly conserved immunodominant heat shock 70 kDa protein of Cryptococcus neoformans var. grubii that covers a considerable digit of the world population through implementing a computational vaccinology approach. MATERIALS AND METHODS: A total of 38 sequences of Cryptococcus neoformans var. grubii's heat shock 70 kDa protein were retrieved from the NCBI protein database. Different prediction tools were used to analyze the aforementioned protein at the Immune Epitope Database (IEDB) to discriminate the most promising T-cell and B-cell epitopes. The proposed T-cell epitopes were subjected to the population coverage analysis tool to compute the global population's coverage. Finally, the T-cell projected epitopes were ranked based on their binding scores and modes using AutoDock Vina software. Results and Discussion. The epitopes (ANYVQASEK, QSEKPKNVNPVI, SEKPKNVNPVI, and EKPKNVNPVI) had shown very strong binding affinity and immunogenic properties to B-cell. (FTQLVAAYL, YVYDTRGKL) and (FFGGKVLNF, FINAQLVDV, and FDYALVQHF) exhibited a very strong binding affinity to MHC-I and MHC-II, respectively, with high population coverage for each, while FYRQGAFEL has shown promising results in terms of its binding profile to MHC-II and MHC-I alleles and good strength of binding when docked with HLA-C∗12:03. In addition, there is massive global population coverage in the three coverage modes. Accordingly, our in silico vaccine is expected to be the future epitope-based peptide vaccine against Cryptococcus neoformans var. grubii that covers a significant figure of the entire world citizens.

CD4+ T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope.

Löfgren's syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3-restricted manner. Using ELISPOT analysis, a greater number of IFN-γ- and IL-2-secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

The macrophage-derived protein PTMA induces filamentation of the human fungal pathogen Candida albicans.

Evasion of killing by immune cells is crucial for fungal survival in the host. For the human fungal pathogen Candida albicans, internalization by macrophages induces a transition from yeast to filaments that promotes macrophage death and fungal escape. Nutrient deprivation, alkaline pH, and oxidative stress have been implicated as triggers of intraphagosomal filamentation; however, the impact of other host-derived factors remained unknown. Here, we show that lysates prepared from macrophage-like cell lines and primary macrophages robustly induce C. albicans filamentation. Enzymatic treatment of lysate implicates a phosphorylated protein, and bioactivity-guided fractionation coupled to mass spectrometry identifies the immunomodulatory phosphoprotein PTMA as a candidate trigger of C. albicans filamentation. Immunoneutralization of PTMA within lysate abolishes its activity, strongly supporting PTMA as a filament-inducing component of macrophage lysate. Adding to the known repertoire of physical factors, this work implicates a host protein in the induction of C. albicans filamentation within immune cells.

Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients.

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

Cryptococcus extracellular vesicles properties and their use as vaccine platforms.

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.