Comparison of virulence between Paracoccidioides brasiliensis and Paracoccidioides lutzii using Galleria mellonella as a host model.

Paracoccidioidomycosis is a systemic mycosis, endemic in Latin America. The etiologic agents of this mycosis are composed of 2 species: Paracoccidioides brasiliensis and P. lutzii. Murine animal models are the gold standard for in vivo studies; however, ethical, economical and logistical considerations limit their use. Galleria mellonella is a suitable model for in vivo studies of fungal infections. In this study, we compared the virulence of P. brasiliensis and P. lutzii in G. mellonella model. The deaths of larvae infected with P. brasiliensis or P. lutzii were similar, and both species were able to reduce the number of hemocytes, which were estimated by microscopy and flow cytometer. Additionally, the phagocytosis percentage was similar for both species, but when we analyze hemocyte-Paracoccidioides spp. interaction using flow cytometer, P. lutzii showed higher interactions with hemocytes. The gene expression of gp43 as well as this protein was higher for P. lutzii, and this expression may contribute to a greater adherence to hemocytes. These results helped us evaluate the behavior of Paracoccidioides spp in G. mellonella, which is a convenient model for investigating the host-Paracoccidioides spp. interaction.

GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus.

The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of ß-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro Galf antigen synthase assay revealed that GfsA has ß1,5- or ß1,6-galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-d-Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal ß-galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O-glycans in the Golgi.

Candida albicans CUG mistranslation is a mechanism to create cell surface variation.

UNLABELLED: In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface ß-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions. IMPORTANCE: The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule ß-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution.

Elevated atmospheric carbon dioxide concentrations amplify Alternaria alternata sporulation and total antigen production.

BACKGROUND: Although the effect of elevated carbon dioxide (CO2) concentration on pollen production has been established in some plant species, impacts on fungal sporulation and antigen production have not been elucidated. OBJECTIVE: Our purpose was to examine the effects of rising atmospheric CO2 concentrations on the quantity and quality of fungal spores produced on timothy (Phleum pratense) leaves. METHODS: Timothy plants were grown at four CO2 concentrations (300, 400, 500, and 600 micromol/mol). Leaves were used as growth substrate for Alternaria alternata and Cladosporium phlei. The spore abundance produced by both fungi, as well as the size (microscopy) and antigenic protein content (ELISA) of A. alternata, were quantified. RESULTS: Leaf carbon-to-nitrogen ratio was greater at 500 and 600 micromol/mol, and leaf biomass was greater at 600 micromol/mol than at the lower CO2 concentrations. Leaf carbon-to-nitrogen ratio was positively correlated with A. alternata spore production per gram of leaf but negatively correlated with antigenic protein content per spore. At 500 and 600 micromol/mol CO2 concentrations, A. alternata produced nearly three times the number of spores and more than twice the total antigenic protein per plant than at lower concentrations. C. phlei spore production was positively correlated with leaf carbon-to-nitrogen ratio, but overall spore production was much lower than in A. alternata, and total per-plant production did not vary among CO2 concentrations. CONCLUSIONS: Elevated CO2 concentrations often increase plant leaf biomass and carbon-to-nitrogen ratio. Here we demonstrate for the first time that these leaf changes are associated with increased spore production by A. alternata, a ubiquitous allergenic fungus. This response may contribute to the increasing prevalence of allergies and asthma.

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG.

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG / Produtos da excreção-secreção e proteases da fase leveduriforme do Sporothrix schenckii: detecção imunológica e clivagem de IgG humana

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

As preparações antigênicas de Sporothrix schenckii provêm geralmente de cultivos mistos de leveduras e micélios e apresentam reações cruzadas com outras micoses profundas. Foi padronizada a obtenção da fase leveduriforme pura, com alto índice de células viáveis, o que permite, por sua vez, obter produtos específicos da excreção-secreção sem contaminantes somáticos. Estes produtos da excreção-secreção são altamente imunogênicos, e não apresentam reações cruzadas visíveis em dupla difusão e sem Western blot. O preparado antigênico consiste principalmente em proteínas com peso molecular entre 40 e 70 kDa, sendo que algumas apresentam atividade proteolítica em meios levemente ácidos. Foi observada atividade do tipo catepsina em produtos da excreção-secreção obtidos a partir de leveduras de dois dias de cultivo, e atividade do tipo quimiotripsina aos quatro dias de cultivo, consistente com a mudança de concentração de proteínas secretadas. As proteases puderam clivar diferentes subclasses de IgG humanas, o que sugere uma produção seqüencial de antígenos e moléculas que podem interagir com a resposta imune do hospedeiro.

Antigen-specific IgE in sinus mucosa of allergic fungal rhinosinusitis patients.

BACKGROUND: Local tissue production of antigen-specific immunoglobulin E (IgE) has been shown in patients with allergic rhinitis and in patients with chronic rhinosinusitis (CRS) with nasal polyps. In allergic fungal rhinosinusitis (AFRS), specific IgE has been established in nasal lavage fluid and eosinophilic mucin. In this study, local production of antigen-specific IgE within sinus mucosa of AFRS patients was evaluated. METHODS: Sinus mucosa homogenates from 11 AFRS patients, 8 patients with CRS without nasal polyps (CRSsNP), and 9 nonrhinosinusitis control patients were assessed for IgE localization by immunohistochemistry. AFRS and control tissue homogenates were also evaluated for antigen-specific IgE to 14 common antigens by ImmunoCAP testing (Phadia AB, Portage, MI). RESULTS: There was a significant increase in IgE staining in AFRS sinus epithelium and subepithelium compared with controls and with patients with CRSsNP (p

Generating cell surface diversity in Candida albicans and other fungal pathogens.

The fungal cell surface contributes to pathogenesis by mediating interactions with host cells and eliciting host immune responses. This review focuses on the cell wall proteome of the major fungal pathogen Candida albicans and discusses how diversity at the cell surface can be introduced by altering the expression and structure of cell wall proteins. Remodelling the cell wall architecture is critical to maintain cellular integrity in response to different environments and stresses including challenge with antifungal drugs. In addition, the dynamic nature of the cell surface alters the physical properties of the fungal interface with host cells and thereby influences adhesion to the host and recognition by components of the host's immune system. Examples of the role of cell surface diversity in the pathogenesis of a number of microorganisms are described.

Use of recombinant gp43 isoforms expressed in Pichia pastoris for diagnosis of paracoccidioidomycosis.

gp43 is the main diagnostic antigen for paracoccidioidomycosis (PCM). In vitro, gp43 expression in supernatant fluids of Paracoccidioides brasiliensis cultures can be unstable, and its regulation is poorly understood. We have been able to express soluble recombinant gp43 (gp43r) isoforms as N-mannosylated proteins secreted in the supernatants of Pichia pastoris cultures induced with methanol. They were secreted as major components from day 2 of induction and could be purified with affinity columns containing anti-gp43 monoclonal antibodies. We have expressed P. brasiliensis GP43 (PbGP43) sequences from genotypes A, D, and E, and the correspondent gp43r isoforms (gp43r A, -B, and -C, respectively; 200 ng) were compared to native gp43 in immunodiffusion (ID) and dot blot assays. Among 90 PCM patient sera showing ID-positive reactions with purified native gp43, 100% were positive with gp43rD and gp43rE and 98% reacted with gp43rA. Of these sera, 78 were tested in dot blot assays at a 1:1,000 dilution, and 100% reacted with all recombinant isoforms. In ID assays, the specificity was 100%, since 40 sera from patients with related mycoses and 30 sera from healthy individuals did not react with any of the antigens. In dot blot assays, 100% specificity for PCM occurred when cross-reactive mannose epitopes were neutralized with 10 mM metaperiodate or eliminated through deglycosylation. However, a 1:1,000 serum dilution was already discriminatory for most sera. We suggest that P. pastoris recombinant gp43, especially isoforms D and E, may replace the native antigen in ID and dot blot assays for diagnosis and prognosis of PCM. Regulated expression of large amounts of antigen in nonpathogenic yeast would justify its preferred use.

Post-antifungal effects of the antifungal compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate on Aspergillus fumigatus.

The post-antifungal effect (PAFE) of the antifungal compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate (DHP) upon Aspergillus fumigatus was investigated. The conidia of A. fumigatus were exposed to DHP at concentrations of 1 x and 4 x MIC(90) for variable times at 37 degrees C. Amphotericin B (AmB)-treated or drug-free controls were included in the study. DHP as well as AmB exposure resulted in prolonged lag phases of the turbidimetric growth curves. Both the treatments gave rise to delayed growth, with lag phases of 11 h upon treatment with a concentration of 4 x MIC(90) for 4 h. Furthermore, it was observed that DHP inhibited the expression of three A. fumigatus secretory proteins of 18, 42 and 55 kDa. One protein of 42 kDa was found to be a metalloprotease, which is an important virulence factor. Analysis of time-dependent antigenic profiles showed the early expression of high-molecular-mass antigens. Expression of low-molecular-mass antigens started after 24 h culture. The antigens of A. fumigatus that are expressed during the early phase of growth were observed to be adversely affected after treatment with DHP. Although the mechanism of action of DHP to inhibit these proteins/antigens is unknown, the observations may be valuable to understand their role in the virulence of the pathogen, as well as the antigen-mediated responses caused by A. fumigatus.

The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis.

Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.

Higher pH level, corresponding to that on the skin of patients with atopic eczema, stimulates the release of Malassezia sympodialis allergens.

BACKGROUND: The opportunistic yeast Malassezia is a trigger factor in atopic eczema (AE). Around 30-80% of patients with AE have an IgE and/or T-cell reactivity to the yeast. Several IgE-binding components have been identified in Malassezia extracts and 11 allergens have been cloned and sequenced. The pH of the skin surface in patients with AE is higher than that of normal healthy skin. We here investigate whether different pH conditions mimicking those of AE skin and healthy skin can influence the production and release of Malassezia allergens. METHODS: Malassezia sympodialis (ATCC strain 42132) was cultured in Dixon broth at pH 6.1 to 5.0 for 1-15 days. Culture supernatants were analysed for the presence of IgE-binding components by immunoblotting. The M. sympodialis cells were analysed for allergen expression and production with immunocytochemistry and quantitative polymerase chain reaction. RESULTS: We found that M. sympodialis cells produce, express and release allergens to a greater extent when cultured at the higher pH. This was particularly true of a 67-kDa major allergen designated Mala s 12. CONCLUSIONS: The data suggest that the skin barrier in AE patients provides an environment that can enhance the release of allergens from M. sympodialis, which can contribute to the inflammation.

Translationally controlled tumor protein from Madurella mycetomatis, a marker for tumorous mycetoma progression.

About 40 years ago Abs against the fungus Madurella mycetomatis were first demonstrated to be present in eumycetoma patients, a disease characterized by tumorous swellings. To date nothing is known about the individual immunoreactive Ags present in this fungus. In the present study, we identify its first immunogenic Ag, a protein homologous to the translationally controlled tumor protein (TCTP), a well-conserved histamine release factor in a range of eukaryotes. The gene for this Ag was demonstrated to be present in two variants in M. mycetomatis, with 13% aa difference between the two proteins encoded. In vitro, TCTP was secreted into the culture medium. In vivo, it was found to be expressed on hyphae present in developing stages of the eumycetoma-characteristic black grain. Significant IgG and IgM immune responses, against the whole protein and selected M. mycetomatis-specific peptides, were determined. The Ab levels correlated with lesion size and disease duration. Overall, the patients with the largest lesions had the highest Ab level, which lowered with decreasing size of the lesion. After 6-15 years of disease duration the Ab levels were the highest. TCTP is the first well-characterized immunogenic Ag, simultaneously the first monomolecular vaccine candidate, identified for the fungus M. mycetomatis.

Characterization of a serodiagnostic complement fixation antigen of Coccidioides posadasii expressed in the nonpathogenic Fungus Uncinocarpus reesii.

Coccidioides spp. (immitis and posadasii) are the causative agents of human coccidioidomycosis. In this study, we developed a novel system to overexpress coccidioidal proteins in a nonpathogenic fungus, Uncinocarpus reesii, which is closely related to Coccidioides. A promoter derived from the heat shock protein gene (HSP60) of Coccidioides posadasii was used to control the transcription of the inserted gene in the constructed coccidioidal protein expression vector (pCE). The chitinase gene (CTS1) of C. posadasii, which encodes the complement fixation antigen, was expressed using this system. The recombinant Cts1 protein (rCts1(Ur)) was induced in pCE-CTS1-transformed U. reesii by elevating the cultivation temperature. The isolated rCts1(Ur) showed chitinolytic activity that was identical to that of the native protein and had serodiagnostic efficacy comparable to those of the commercially available antigens in immunodiffusion-complement fixation tests. Using the purified rCts1(Ur), 74 out of the 77 coccidioidomycosis patients examined (96.1%) were positively identified by enzyme-linked immunosorbent assay. The rCts1(Ur) protein showed higher chitinolytic activity and slightly greater seroreactivity than the bacterially expressed recombinant Cts1. These data suggest that this novel expression system is a useful tool to produce coccidioidal antigens for use as diagnostic antigens.

Morphological, biochemical and molecular approaches for comparing typical and atypical Paracoccidioides brasiliensis strains.

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein.

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.

Virulence of Paracoccidioides brasiliensis and gp43 expression in isolates bearing known PbGP43 genotype.

Paracoccidioides brasiliensis is the dimorphic fungus responsible for human paracoccidioidomycosis (PCM). We previously observed that P. brasiliensis isolates bearing highly polymorphic PbGP43 of genotype A (Pb2, Pb3 and Pb4) were phylogenetically distant from the others. The PbGP43 gene encodes an immune dominant diagnostic antigen (gp43), and its polymorphism reflects broader genetic diversity in the species. In the present study, we observed that isolates with PbGP43 of genotype A showed low virulence when inoculated in B10.A mice by the intraperitoneal, intratracheal and intravenous routes. In vitro studies detected sharp and prolonged down-regulation of PbGP43 in Pb3 (and not in Pb18 or Pb339) as a result of heat shock at 42 degrees C and temperature shift to prompt mycelium to yeast transition, which was, however, not disturbed. Differences in transcriptional regulation are possibly a consequence of mutations in the PbGP43 promoter region, which we here show to be more polymorphic in genotype A isolates. As opposed to Pb3's rapid adaptation to in vitro culture conditions after isolation from the lung, Pb12, the most aggressive isolate tested here, showed slow growth and phase transition in vitro. Interestingly, animals that were highly infected by Pb12 produced small amounts of anti-gp43 antibodies. That was apparently due to down-regulation in PbGP43 expression. We present the first evidence of transcriptional regulation of gp43 expression, but our results suggest that gene expression is also regulated at the protein and/or secretion levels.

Production profile of the major allergen Alt a 1 in Alternaria alternata cultures.

BACKGROUND: The fungus Alternaria is strongly associated with asthma, but the importance of fungal allergen products is frequently underestimated. The profile of allergen release from fungal material is poorly understood. OBJECTIVE: To investigate expression of the major allergen of Alternaria alternata, Alt a 1, during its growth in culture conditions for allergen extract production. METHODS: Allergen expression was examined by Alt a 1-specific 2-site monoclonal antibody enzyme-linked immunosorbent assay, immunoblotting, and potency assays. The release of Alt a 1 was studied by transmission electron microscopy in conjunction with immunogold staining by using antibodies with specificity for Alt a 1. RESULTS: A maximum amount of Alt a 1 was obtained after 4.5 weeks of growing, and it was found predominantly in the spent culture medium. In the same way, total IgE binding activity showed 15-fold more activity in the spent culture medium than in the buffer-extractable antigen fraction. Immunogold electron microscopy provided evidence that Alt a 1 is released from spores and mycelia. CONCLUSIONS: Alternaria alternata allergenic proteins were constantly released into the culture medium, where they accumulated. Alt a 1 was a good marker for checking optimal culture conditions for A alternata extract production intended for clinical use.

Cholera vaccine candidate 638: intranasal immunogenicity and expression of a foreign antigen from the pulmonary pathogen Coccidioides immitis.

Vibrio cholerae strain 638 is a live genetically attenuated candidate cholera vaccine in which the CTXPhi prophage encoding cholera toxin has been deleted and hapA, encoding an extracellular Zn-dependent metalloprotease, was insertionally inactivated. Strain 638 was highly immunogenic when inoculated to adult Swiss mice by the intranasal route as judged by the induction of a strong serum vibriocidal antibody response. A side-by-side comparison of strain 638 with its isogenic hapA(+) precursor (strain 81) in the above model indicated that inactivation of hapA does not affect immunogenicity. The spherule-associated antigen 2/proline-rich antigen (Ag2/PRA) of Coccidioides immitis has been shown to protect mice against coccidioidomycosis to an extent dependent on the modes of antigen presentation and challenge with C. immitis arthrospores. In this work, we demonstrate the use of a live genetically attenuated V. cholerae strain to deliver Ag2/PRA. Ag2/PRA was expressed in 638 as a fusion protein with the Escherichia coli heat labile toxin B subunit leader peptide using the strong Tac promoter. The recombinant Ag2/PRA was efficiently expressed, processed and secreted to the periplasmic space. Intranasal immunizations of adult mice with strain 638 expressing Ag2/PRA induced serum vibriocidal antibody response to the vector strain and serum total IgG response to Ag2/PRA. Strain 638 expressing PRA could be recovered from trachea and lung up to 20h after immunization but was effectively cleared 72h post-inoculation.

Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris.

BP230 is a cytoskeletal linker protein of 2649 amino acids originally identified as the target autoantigen in bullous pemphigoid, a potentially devastating autoimmune skin blistering disorder. To better define its function, we sought to generate recombinant forms of BP230 in both Saccharomyces cerevisiae and Pichia pastoris after cloning its entire cDNA. By immunoblot analysis, full-length BP230 was not found in extracts of P. pastoris, whereas minor amounts of degraded BP230 were detected in extracts of S. cerevisiae. In contrast, both S. cerevisiae and P. pastoris were able to produce the 770-amino acid COOH-terminal domain of BP230. Furthermore, the production level of the recombinant BP230 tail in S. cerevisiae was significantly higher than that observed in P. pastoris and that of endogenous BP230 in cultured human keratinocytes. Finally, 12 of 17 (71%) BP sera recognized the recombinant BP230 protein in yeast extracts. Our results indicate that S. cerevisiae occasionally constitutes a better tool for recombinant protein production than P. pastoris. Although both its large size and poor solubility limit production of BP230, the developed yeast system provides cellular fractions enriched in BP230 recombinant proteins that constitute useful tools for the diagnosis of bullous pemphigoid.