Cryptococcus neoformans Capsular GXM Conformation and Epitope Presentation: A Molecular Modelling Study.

The pathogenic encapsulated Cryptococcus neoformans fungus causes serious disease in immunosuppressed hosts. The capsule, a key virulence factor, consists primarily of the glucuronoxylomannan polysaccharide (GXM) that varies in composition according to serotype. While GXM is a potential vaccine target, vaccine development has been confounded by the existence of epitopes that elicit non-protective antibodies. Although there is evidence for protective antibodies binding conformational epitopes, the secondary structure of GXM remains an unsolved problem. Here an array of molecular dynamics simulations reveal that the GXM mannan backbone is consistently extended and relatively inflexible in both C. neoformans serotypes A and D. Backbone substitution does not alter the secondary structure, but rather adds structural motifs: ß DGlcA and ß DXyl side chains decorate the mannan backbone in two hydrophillic fringes, with mannose-6-O-acetylation forming a hydrophobic ridge between them. This work provides mechanistic rationales for clinical observations-the importance of O-acetylation for antibody binding; the lack of binding of protective antibodies to short GXM fragments; the existence of epitopes that elicit non-protective antibodies; and the self-aggregation of GXM chains-indicating that molecular modelling can play a role in the rational design of conjugate vaccines.

Interaction of Alt a 1 with SLC22A17 in the airway mucosa.

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.

ß-1,6-linked Galactofuranose- rich peptidogalactomannan of Fusarium oxysporum is important in the activation of macrophage mechanisms and as a potential diagnostic antigen.

A peptidogalactomannan (PGM) from Fusarium oxysporum was structurally characterized by a combination of chemical and spectroscopic methods, including one and two-dimensional nuclear magnetic resonance (1D and 2D NMR). The galactomannan component consists of a main chain containing (1→6)-linked ß-D-galactofuranose residues with side chains containing (1→2)-linked α-D-Glcp, (1→2)-linked -ß-D-Manp (1→2) and ß-D-Manp terminal nonreducing end units and differs from that of Aspergillus fumigatus and Cladosporium resinae that present a main chain containing (1→6)-linked α-D-Manp residues presenting ß-D-Galf as side chains of 3-4 units that are (1→5)-interlinked. The importance of the carbohydrate moiety of the F. oxysporum PGM was demonstrated. Periodate oxidation abolished much of the PGM antigenic activity. A strong decrease in reactivity was also observed with de-O-glycosylated PGM. In addition, de-O-glycosylated PGM was not able to inhibit F. oxysporum phagocytosis, suggesting that macrophages recognize and internalize F. oxysporum via PGM. F. oxysporum PGM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PGM led to a significant increase of TNF-α cytokine levels, suggesting that their removal could exposure another PGM motifs able to induce a higher secretion of TNF-α levels. Interestingly, F. oxysporum conidia, intact and de-O-linked PGM were not able to induce IL-10 cytokine release. The difference in patient serum reativity using a PGM from F. oxysporum characterized in the present study as compared with a PGM from C. resinae, that presents the same epitopes recognized by serum from patients with aspergillosis, could be considered a potential diagnostic antigen and should be tested with more sera.

Analysis of the 3H8 antigen of Candida albicans reveals new aspects of the organization of fungal cell wall proteins.

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 ß-1,3-glucanase, ß mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall ß-1,3 glucans, and to proteins by disulfide bonds, but not to ß-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 ß 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.

Selection of Specific Peptides for Coccidioides spp. Obtained from Antigenic Fractions through SDS-PAGE and Western Blot Methods by the Recognition of Sera from Patients with Coccidioidomycosis.

Antigenic fractions of 100, 50, 37, and 28 kDa obtained through the SDS-PAGE method that were more frequently recognized by anti-Coccidioides antibodies in the sera of coccidioidomycosis patients were selected using western blotting. Subsequently, these bands were sequenced, and the obtained proteins were analysed by BLAST to choose peptides specific for Coccidioides spp. from among the shared aligned sequences of related fungi. A peptide specific for C. immitis was selected from the "GPI anchored serine-threonine rich protein OS C. immitis", while from the "uncharacterized protein of C. immitis", we selected a peptide for C. immitis and C. posadasii. These proteins arose from the 100 kDa antigenic fraction. From the protein "fatty acid amide hydrolase 1 of C. posadasii" that was identified from the 50 kDa antigenic fraction, a peptide was selected that recognized C. immitis and C. posadasii. In addition, the analysis of all the peptides (353) of each of the assembled proteins showed that only 35 had 100% identity with proteins of C. immitis and C. posadasii, one had 100% identity with only C. immitis, and one had 100% identity with only C. posadasii. These peptides can be used as diagnostic reagents, vaccines, and antifungals.

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-ß) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.

[ON DEVELOPMENT OF TOOLS OF IMMUNE DIAGNOSTIC OF INFECTIOUS DISEASES: PROBLEMS OF DIAGNOSTIC IN VIVO AND IN VITRO].

The article considers a number of problematic issues concerning development of effective means of immune diagnostic of infectious diseases of bacterial and mycotic etiology related to approaches of choosing appropriate diagnostic targets.

Significant structural change in both O- and N-linked carbohydrate moieties of the antigenic galactomannan from Aspergillus fumigatus grown under different culture conditions.

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 ß-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.

Structure and function of a fungal adhesin that binds heparin and mimics thrombospondin-1 by blocking T cell activation and effector function.

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.

Synthesis and immunological studies of linear oligosaccharides of ß-glucan as antigens for antifungal vaccine development.

Antifungal vaccines have recently engendered considerable excitement for counteracting the resurgence of fungal infections. In this context, ß-glucan, which is abundantly expressed on all fungal cell surfaces, functionally necessary for fungi, and immunologically active, is an attractive target antigen. Aiming at the development of effective antifungal vaccines based on ß-glucan, a series of its oligosaccharide derivatives was designed, synthesized, and coupled with a carrier protein, keyhole limpet hemocyanin (KLH), to form new semisynthetic glycoconjugate vaccines. In this article, a convergent and effective synthetic strategy using preactivation-based iterative glycosylation was developed for the designed oligosaccharides. The strategy can be widely useful for rapid construction of large oligo-ß-glucans with shorter oligosaccharides as building blocks. The KLH conjugates of the synthesized ß-glucan hexa-, octa-, deca-, and dodecasaccharides were demonstrated to elicit high titers of antigen-specific total and IgG antibodies in mice, suggesting the induction of functional T cell-mediated immunity. Moreover, it was revealed that octa-, deca-, and dodeca-ß-glucans were much more immunogenic than the hexamer and that the octamer was the best among these. The results suggested that the optimal oligosaccharide sequence of ß-glucan required for exceptional immunogenicity was a hepta- or octamer and that longer glucans are not necessarily better antigens, a finding that may be of general importance. Most importantly, the octa-ß-glucan-KLH conjugate provoked protective immunity against Candida albicans infection in a systemic challenge model in mice, suggesting the great potential of this glycoconjugate as a clinically useful immunoprophylactic antifungal vaccine.

Charge-based engineering of hydrophobin HFBI: effect on interfacial assembly and interactions.

Hydrophobins are extracellular proteins produced by filamentous fungi. They show a variety of functions at interfaces that help fungi to adapt to their environment by, for example, adhesion, formation of coatings, and lowering the surface tension of water. Hydrophobins fold into a globular structure and have a distinct hydrophobic patch on their surface that makes these proteins amphiphilic. Their amphiphilicity implies interfacial assembly, but observations indicate that intermolecular interactions also contribute to their functional properties. Here, we used the class II hydrophobin HFBI from Trichoderma reesei as a model to understand the structural basis for the function of hydrophobins. Four different variants were made in which charged residues were mutated. The residues were chosen to probe the role of different regions of the hydrophilic part of the proteins. Effects of the mutations were studied by analyzing the formation and structure of self-assembled layers, multimerization in solution, surface adhesion, binding of secondary layers of proteins on hydrophobins, and the viscoelastic behavior of the air-water interface during formation of protein films; the comparison showed clear differences between variants only in the last two analyses. Surface viscoelasticity behavior suggests that the formation of surface layers is regulated by specific interactions that lead to docking of proteins to each other. One set of mutations led to assemblies with a remarkably high elasticity at the air-water interface (1.44 N/m). The variation of binding of secondary layers of protein on surface-adsorbed hydrophobins suggest a mechanism for a proposed function of hydrophobins, namely, that hydrophobins can act as a specific adhesive layer for the binding of macromolecules to interfaces.

Surface hydrophobin prevents immune recognition of airborne fungal spores.

The air we breathe is filled with thousands of fungal spores (conidia) per cubic metre, which in certain composting environments can easily exceed 10(9) per cubic metre. They originate from more than a hundred fungal species belonging mainly to the genera Cladosporium, Penicillium, Alternaria and Aspergillus. Although these conidia contain many antigens and allergens, it is not known why airborne fungal microflora do not activate the host innate immune cells continuously and do not induce detrimental inflammatory responses following their inhalation. Here we show that the surface layer on the dormant conidia masks their recognition by the immune system and hence prevents immune response. To explore this, we used several fungal members of the airborne microflora, including the human opportunistic fungal pathogen Aspergillus fumigatus, in in vitro assays with dendritic cells and alveolar macrophages and in in vivo murine experiments. In A. fumigatus, this surface 'rodlet layer' is composed of hydrophobic RodA protein covalently bound to the conidial cell wall through glycosylphosphatidylinositol-remnants. RodA extracted from conidia of A. fumigatus was immunologically inert and did not induce dendritic cell or alveolar macrophage maturation and activation, and failed to activate helper T-cell immune responses in vivo. The removal of this surface 'rodlet/hydrophobin layer' either chemically (using hydrofluoric acid), genetically (DeltarodA mutant) or biologically (germination) resulted in conidial morphotypes inducing immune activation. All these observations show that the hydrophobic rodlet layer on the conidial cell surface immunologically silences airborne moulds.

A monoclonal antibody against 47.2 kDa cell surface antigen prevents adherence and affects biofilm formation of Candida albicans.

Candida albicans is an opportunistic dimorphic pathogen that exists in both planktonic and biofilm phases causing deep-rooted infections in mainly immunocompromised patients. Antibodies are believed to play anti-Candida activity by different mechanisms, like inhibition of adhesion and neutralization of virulence-related antigens. Inhibition of adhesion is one of the important strategies to prevent Candida infections and biofilm formation. In this study, monoclonal antibody (MAb 7D7) against C. albicans biofilm cell surface antigen (47.2 kDa) was generated to determine the changes in adherence and viability of C. albicans. In this regard XTT assay was carried out in 30, 60, 90 min and 48 h (maturation time) time points using MAb 7D7 and it (MAb 7D7) was found to be effective against adhesion and the formation of C. albicans biofilm on polystyrene as well as monolayer of human epithelial cells (HeLa). This result may also prove to be a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells.

[Demonstration of ß-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies]. / Candida albicans maya hücre duvarinda eksprese edilip hif duvarinda edilmeyen ß-1,2 mannan yapilarinin monoklonal antikorlar ile gösterilmesi.

Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method. Sandwich ELISA and immunofluorescence (IF) methods have been used to detect the experimental reactions. In our study, highly specific class IgM murine monoclonal antibodies (mAb-2B7) against C.albicans yeast cell wall were obtained from clone 2B7. These antibodies cross-reacted with S.choleraesuis 211 and S.infantis bacteria sharing similar cell wall structure of C.albicans. The existence of mannan ß-1,2 bonds on the surface of C.albicans yeast form was confirmed with a commercial monoclonal antibody (mAb-ACMK-1; Matriks Biotek(®), Turkey) specific for those bonds. Besides, mAb-ACMK-1 interacted with C.albicans yeast form and gave intense fluorescence (high positive reaction) in IF method, but no fluorescence (negative) was detected with hyphal form. This data, obtained for the first time with this study, indicates that the mannan ß-1,2 bonds are either found infrequently or none in the fungal hyphal wall. Although both monoclonal antibodies recognize the mannan antigen, mAb-2B7 reacted with S.choleraesuis 211, while mAb-ACMK-1 did not, due to the difference of epitope specificity. In conclusion, monoclonal antibodies may facilitate the characterization of antigenic structures of Candida, which will lead for the identification of new determinants that may increase the sensitivity and specificity of commercial tests used for mannan detection in serum.

Distinct and complementary roles for Aspergillus fumigatus-specific Tr1 and Foxp3+ regulatory T cells in humans and mice.

Unlike induced Foxp3(+) regulatory T cells (Foxp3(+) iTreg) that have been shown to play an essential role in the development of protective immunity to the ubiquitous mold Aspergillus fumigatus, type-(1)-regulatory T cells (Tr1) cells have, thus far, not been implicated in this process. Here, we evaluated the role of Tr1 cells specific for an epitope derived from the cell wall glucanase Crf-1 of A. fumigatus (Crf-1/p41) in antifungal immunity. We identified Crf-1/p41-specific latent-associated peptide(+) Tr1 cells in healthy humans and mice after vaccination with Crf-1/p41+zymosan. These cells produced high amounts of interleukin (IL)-10 and suppressed the expansion of antigen-specific T cells in vitro and in vivo. In mice, in vivo differentiation of Tr1 cells was dependent on the presence of the aryl hydrocarbon receptor, c-Maf and IL-27. Moreover, in comparison to Tr1 cells, Foxp3(+) iTreg that recognize the same epitope were induced in an interferon gamma-type inflammatory environment and more potently suppressed innate immune cell activities. Overall, our data show that Tr1 cells are involved in the maintenance of antifungal immune homeostasis, and most likely play a distinct, yet complementary, role compared with Foxp3(+) iTreg.

Glucoamylase is a major allergen of Schizophyllum commune.

BACKGROUND: Schizophyllum commune is one of the causative agents of basidiomycosis including disorders such as allergic bronchopulmonary mycosis, allergic fungal sinusitis, and mucoid impaction of bronchi, the incidence of those of which has been increasing. These mycoses are difficult to diagnose because only a limited number of diagnostic tools are currently available. The biggest problem is that no specific antigens of S. commune have been identified to enable serodiagnosis of the disease. OBJECTIVE: In this study, we attempted to identify a major antigen of S. commune to establish a reliable serodiagnostic method. METHODS: We used mass spectrometry to identify an antigen that reacted with the serum of a patient with allergic bronchopulmonary mycosis caused by S. commune. The protein was expressed in Escherichia coli, highly purified, and the patient sera IgG and IgE titres against the protein were determined by enzyme-linked immunosorbent assay. RESULTS: The protein identified as a major antigen of S. commune was named Sch c 1; it was a homolog of glucoamylase. The IgG and IgE titres against Sch c 1 in patient sera were significantly higher than those in healthy volunteer sera (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Sch c 1 is recognized by the host immune system of patients as an antigen/allergen. The purified glucoamylase Sch c 1 is a promising candidate antigen for the serodiagnosis of S. commune-induced mycosis.

TLR15 is unique to avian and reptilian lineages and recognizes a yeast-derived agonist.

The TLRs represent a family of pattern recognition receptors critical in the induction of vertebrate immune responses. Between 10 and 13 different TLR genes can be identified in each vertebrate species, with many represented as orthologous genes in different species. The agonist specificity of orthologous TLR is also highly conserved. In contrast, TLR15 can only be identified in avian and reptilian genomes, suggesting that this receptor arose ~320 million years ago after divergence of the bird/reptile and mammalian lineages. Transfection of a constitutively active form of chicken TLR15 led to NF-κB activation in HEK293 cells and induced cytokine mRNA upregulation in chicken cell lines. Full-length TLR15 mediated NF-κB induction in response to lysates from yeast, but not those derived from viral or bacterial pathogens, or a panel of well-characterized TLR agonists. TLR15 responses were induced by whole-cell lysates derived from Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, but not zymosan preparations from S. cerevisiae. The ability of yeast lysate to activate TLR15-dependent NF-κB pathways (in transfection assays) or stimulate IL-1ß mRNA upregulation in chicken macrophages was abrogated by heat inactivation or pre-exposure of the lysate to PMSF. Identification of yeast as an agonist source for TLR15 provides a functional framework for consideration of this TLR within the context of pattern recognition receptor evolution and may impact on the development of novel adjuvants.

Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins.

Class I fungal hydrophobins are small surface-active proteins that self-assemble to form amphipathic monolayers composed of amyloid-like rodlets. The monolayers are extremely robust and can adsorb onto both hydrophobic and hydrophilic surfaces to reverse their wettability. This adherence is particularly strong for hydrophobic materials. In this report, we show that the class I hydrophobins EAS and HYD3 can self-assemble to form a single-molecule thick coating on a range of nanomaterials, including single-walled carbon nanotubes (SWCNTs), graphene sheets, highly oriented pyrolytic graphite, and mica. Moreover, coating by class I hydrophobin results in a stable, dispersed preparation of SWCNTs in aqueous solutions. No cytotoxicity is detected when hydrophobin or hydrophobin-coated SWCNTs are incubated with Caco-2 cells in vitro. In addition, we are able to specifically introduce covalently linked chemical moieties to the hydrophilic side of the rodlet monolayer. Hence, class I hydrophobins provide a simple and effective strategy for controlling the surfaces of a range of materials at a molecular level and exhibit strong potential for biomedical applications.

Characterization of an antigenic chitosanase from the cellulolytic fungus Chaetomium globosum.

We are interested in identifying human fungal allergens and antigens from species common on water-damaged or damp building materials for use as marker proteins and diagnostic tests. The cellulolytic fungus Chaetomium globosum is common on damp materials in the building environment worldwide. ELISA and immunoblotting tests identified two related proteins of molecular weights 45 and 47 kDa which were identified as fungal antigens found on spore surfaces and in culture filtrate. The sequences were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS), which indicated that the two proteins were chitosanases, confirmed by enzyme assay. The 47 kDa protein was not glycosylated and had an acidic pI of 4.5. These proteins have not been reported from other fungi and similar antigens were not seen in other fungi common in buildings. The production of polyclonal antibodies in rabbits showed the antigenicity of the target proteins and confirmed they were not artifacts of the isolation process. The proteins isolated are useful biomarkers for the detection of C. globosum in the building environment.

Chitin-like molecules associate with Cryptococcus neoformans glucuronoxylomannan to form a glycan complex with previously unknown properties.

In prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-α). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties.