Chloroquine modulates the fungal immune response in phagocytic cells from patients with chronic granulomatous disease.

Invasive aspergillosis is a major threat to patients with chronic granulomatous disease (CGD). Fungal pathogenesis is the result of a diminished antifungal capacity and dysregulated inflammation. A deficient NADPH-oxidase complex results in defective phagolysosomal alkalization. To investigate the contribution of defective pH regulation in phagocytes among patients with CGD during fungal pathogenesis, we evaluated the effect of the acidotropic, antimalarial drug chloroquine (CQ) on the antifungal capacity of polymorphonuclear cells (PMNs) and on the inflammatory response of peripheral blood mononuclear cells (PBMCs). Chloroquine exerted a direct pH-dependent antifungal effect on Aspergillus fumigatus and Aspergillus nidulans; it increased the antifungal activity of PMNs from patients with CGD at a significantly lower concentration, compared with the concentration for PMNs from healthy individuals; and decreased the hyperinflammatory state of PBMCs from patients with CGD, as observed by decreased tumor necrosis factor α and interleukin 1ß release. Chloroquine targets both limbs of fungal pathogenesis and might be of great value in the clearance of invasive aspergillosis in patients with CGD.

Human leukocytes kill Aspergillus nidulans by reactive oxygen species-independent mechanisms.

Invasive aspergillosis is a major threat for patients suffering from chronic granulomatous disease (CGD). Although Aspergillus fumigatus is the most commonly encountered Aspergillus species, the presence of A. nidulans appears to be disproportionately high in CGD patients. The purpose of this study was to investigate the involvement of the NADPH oxidase and the resulting reactive oxygen species (ROS) in host defense against fungi and to clarify their relationship toward A. nidulans. Murine CGD alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from healthy controls and CGD patients were challenged with either A. fumigatus or A. nidulans. Analysis of the antifungal effects of ROS revealed that A. nidulans, in contrast to A. fumigatus, is not susceptible to ROS. In addition, infection with live A. nidulans did not result in any measurable ROS release. Remarkably, human CGD PMN and PBMC and murine CGD AM were at least equipotent at arresting conidial germination compared to healthy controls. Blocking of the NADPH oxidase resulted in significantly reduced damage of A. fumigatus but did not affect A. nidulans hyphae. Furthermore, the microbicidal activity of CGD PMN was maintained toward A. nidulans but not A. fumigatus. In summary, antifungal resistance to A. nidulans is not directly ROS related. The etiology of A. nidulans infections in CGD cannot be explained by the simple absence of the direct microbicidal effect of ROS. In vivo, the NADPH oxidase is a critical regulator of innate immunity whose unraveling will improve our understanding of fungal pathogenesis in CGD.

Experimental evolution of defense against a competitive mold confers reduced sensitivity to fungal toxins but no increased resistance in Drosophila larvae.

BACKGROUND: Fungal secondary metabolites have been suggested to function as chemical defenses against insect antagonists, i.e. predators and competitors. Because insects and fungi often compete for dead organic material, insects may achieve protection against fungi by reducing sensitivity to fungal chemicals. This, in turn, may lead to increased resistance allowing insects better to suppress the spread of antagonistic but non-pathogenic microbes in their habitat. However, it remains controversial whether fungal toxins serve as a chemical shield that selects for insects that are less sensitive to toxins, and hence favors the evolution of insect resistance against microbial competitors. RESULTS: To examine the relationship between the ability to survive competition with toxic fungi, sensitivity to fungal toxins and resistance, we created fungal-selected (FS) replicated insect lines by exposing Drosophila melanogaster larvae to the fungal competitor Aspergillus nidulans over 26 insect generations. Compared to unselected control lines (UC), larvae from the FS lines had higher survival rates in the presence of A. nidulans indicating selection for increased protection against the fungal antagonist. In line with our expectation, FS lines were less susceptible to the A. nidulans mycotoxin Sterigmatocystin. Of particular interest is that evolved protection against A. nidulans and Sterigmatocytin was not correlated with increased insect survival in the presence of other fungi and mycotoxins. We found no evidence that FS lines were better at suppressing the expansion of fungal colonies but observed a trend towards a less detrimental effect of FS larvae on fungal growth. CONCLUSION: Antagonistic but non-pathogenic fungi favor insect variants better protected against the fungal chemical arsenal. This highlights the often proposed but experimentally underexplored importance of secondary metabolites in driving animal-fungus interactions. Instead of enhanced resistance, insect larvae tend to have evolved increased tolerance of the fungal competitor. Future studies should examine whether sensitivity to allelopathic microbial metabolites drives a trade-off between resistance and tolerance in insect external defense.

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.

Experimental evolution of resistance against a competing fungus in Drosophila melanogaster.

Competition between microorganisms and arthropods has been shown to be an important ecological interaction determining animal development and spatial distribution patterns in saprophagous communities. In fruit-inhabiting Drosophila, variation in insect developmental success is not only determined by species-specific effects of various noxious filamentous fungi but, as suggested by an earlier study, also by additive genetic variation in the ability to successfully withstand the negative impact of the fungi. If this variation represents a direct adaptive response to the degree to which insect breeding substrates are infested with harmful fungi, genetic variation for successful development in the presence of fungi could be maintained by variation in infestation of resource patches with fungi. We selected for the ability to resist the negative influence of mould by maintaining replicated Drosophila melanogaster populations on substrates infested with Aspergillus nidulans. After five cycles of exposure to the fungus during the larval stage, the selected populations were compared with unselected control populations regarding adult survival and reproduction to reveal an evolved resistance against the fungal competitor. On fungus-infested larval feeding substrates, emerged adults from mould-selected populations had higher survival rates and higher early fecundity than the control populations. In the unselected populations, females had higher mortality rates than males, and a high proportion of both females and males appeared to be unable to lay eggs or fertilise eggs, respectively. When larvae developed on non-infested food we found indications of a loss of resistance to abiotic and starvation stress in the adult stage in flies from the selected populations. This suggests that there are costs associated with an increase in resistance against the microbial competitor. We discuss the underlying mechanisms that might have selected for increased resistance against harmful fungi.

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production.

AIMS: Elucidation of the regulation of ChiB production in Aspergillus nidulans. METHODS AND RESULTS: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. CONCLUSIONS: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.

Differential Kinetics of Aspergillus nidulans and Aspergillus fumigatus Phagocytosis.

Invasive aspergillosis mainly occurs in immunocompromised patients and is commonly caused by Aspergillus fumigatus, while A.nidulans is rarely the causative agent. However, in chronic granulomatous disease (CGD) patients, A. nidulans is a frequent cause of invasive aspergillosis and is associated with higher mortality. Immune recognition of A. nidulans was compared to A. fumigatus to offer an insight into why A. nidulans infections are prevalent in CGD. Live cell imaging with J774A.1 macrophage-like cells and LC3-GFP-mCherry bone marrow-derived macrophages (BMDMs) revealed that phagocytosis of A. nidulans was slower compared to A. fumigatus. This difference could be attributed to slower migration of J774A.1 cells and a lower percentage of migrating BMDMs. In addition, delayed phagosome acidification and LC3-associated phagocytosis was observed with A. nidulans. Cytokine and oxidative burst measurements in human peripheral blood mononuclear cells revealed a lower oxidative burst upon challenge with A. nidulans. In contrast, A. nidulans induced significantly higher concentrations of cytokines. Collectively, our data demonstrate that A. nidulans is phagocytosed and processed at a slower rate compared to A. fumigatus, resulting in reduced fungal killing and increased germination of conidia. This slower rate of A. nidulans clearance may be permissive for overgrowth within certain immune settings.

[LTR retrotransposons from genomes of Aspergillus fumigatus and A. nidulans].

Fungi Aspergillus spp. are able to infect all tissues and organs and often cause invasive mycosis (aspergillosis), which is usually a fatal disease, especially in the patients with compromised immune system. Microbiological monitoring of these infectious agents is necessary in modem medical facilities. Mobile elements can be used as markers for identification of species and strains of Aspergillus found indoors as well as in aspergillosis diagnostics. Genomic sequences of two representative Aspergillus species, A. fumigatus and A. nidulans, were analysed in silico in order to detect LTR retrotransposons. We found considerable differences in the composition of retrotransposon families between two studied species. One of the detected families, which is present in both studied Aspergillus species, is phylogenetically quite different from all other known fungal retrotransposons. The majority of elements are represented by damaged copies. Nevertheless, we describe for the first time allegedly non-damaged LTR copies that contain intact ORFs and could be active.

Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant ß1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.

Aspergillus species cystitis in a cat.

A Persian male cat with a history of lower urinary tract disease was presented because of polydipsia, polyuria, constipation and nasal discharge. Ten weeks before admission, the cat had been treated for lower urinary tract disease by catheterisation and flushing of the bladder. The animal was thin, dehydrated, anaemic and azotaemic. Urine culture revealed Aspergillus species cystitis. Antibodies against Aspergillus nidulans were identified in serum. Fluconazole was administered orally (7.5 mg/kg, q 12 h) for 10 consecutive weeks. The azotaemia was resolved, the kidney concentrating ability was recovered and the cat has remained healthy without similar problems.

[RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response]. / Bialka regulujace przekazywanie sygnalu przez bialka G (bialka RGS) i ich znaczenie w regulacji odpowiedzi immunologicznej.

RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins. By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction. Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins). The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies. In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways. They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation. They also take an essential part in inflammation. High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors. The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response

[Relation of bronchial asthma and allergic bronchopulmonary aspergillosis]. / O sootnoshenii bronkhial'noi astmy i allergicheskogo bronkholegochenogo aspergilleza.

An allergological examination was conducted in 30 out of 83 (36.07%) patients showing symptoms of bronchial asthma. It was found that skin hypersensitivity to Aspergilla allergens coupled with a high serum IgE level. The appropriateness of such affection referral to a separate nosological entity (allergic bronchopulmonary aspergillosis) is under discussion.

Induced fungal resistance to insect grazing: reciprocal fitness consequences and fungal gene expression in the Drosophila-Aspergillus model system.

BACKGROUND: Fungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding. CONCLUSIONS/SIGNIFICANCE: Our study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in fungivore-rich habitats. Induced fungal defence responses thus need to be included if we wish to have a complete conception of animal-fungus co-evolution, fungal gene regulation, and multitrophic interactions.

Virulence comparisons of Aspergillus nidulans mutants are confounded by the inflammatory response of p47phox-/- mice.

While investigating the requirement for phagosomal alkalinization in the host defense against pulmonary aspergillosis, we observed high morbidity of p47(phox)(-/-) mice infected with pH-insensitive Aspergillus nidulans mutants despite a paucity of fungal growth. Fatal infection also resulted from a normally avirulent p-aminobenzoate auxotroph. This demonstrates that p47(phox)(-/-) murine immunity contributes significantly to A. nidulans lethality. These data have wider implications for microbial virulence studies with p47(phox)(-/-) mice.

Immunogenic potential of Aspergillus nidulans subcellular fractions and their polypeptide components.

Cell-free extracts of the ascomycetous fungus Aspergillus nidulans were separated into three subcellular fractions: cell walls, total membranes and cytosol, and two different immunization protocols were used to raise antibodies against them in 12 New Zealand rabbits. The immune response was followed over time by dot and Western blot analyses to determine the immunogenic potential of each individual fraction and their polypeptide components. The IgG fractions, purified from pools of the best sera, were used to analyze in detail the antigenic composition of A. nidulans mycelium. The fast immunization protocol provided a much earlier response and higher sera titres. Cytosols and membranes were more immunogenic than cell walls and, in most cases, a positive correlation was shown between the titre of each serum and the number of detected antigens. The polypeptides of A. nidulans included six major immunodominant antigens of the molecular weights ranging between 13 and 200 kDa.

Variability of Aspergillus nidulans antigens with media and time and temperature of growth.

The influence of culture medium and time and temperature of growth on the appearance of Aspergillus nidulans antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or Western blot (immunoblot), of the proteins present in total cellular extracts or culture supernatants. Samples in the exponential, deceleration, and stationary growth phases were selected by biochemical, morphological, and ultrastructural criteria. Protein and antigen patterns (detected with rabbit antibodies) from total extracts were very similar in all cases, and the major differences observed seemed to depend on the age of the cultures. Culture supernatant patterns were highly dependent on the type of medium (complex or defined) and the age of the culture. Temperature did not significantly influence these results. The reproducible reactivity of selected human sera from aspergilloma-affected individuals was strictly associated with the use of defined media, especially Czapek Dox-AOAC, in both total extracts and culture supernatants. Extended growth times were necessary in the case of metabolic antigens (those obtained from culture supernatants). Screening of a battery of 10 selected human serum samples from patients with aspergilloma or invasive aspergillosis demonstrated that two of the antigens (96 to 98 and 45 kDa) from stationary-phase culture supernatants in Czapek Dox-AOAC medium were consistently reactive. When considered together as one unit, both antigens reacted with more than 50% of the sera, and at least one or the other of the antigens reacted with more than 90% of the sera. Less consistent results were obtained for two somatic antigens (from total cell extracts) of 45 to 50 and 20 to 22 kDa.

The preparation and partial characterization of antigenic fractions obtained from the mycelial walls of several Aspergillus species.

Extracts with immunological activity were prepared from Aspergillus fumigatus, A. flavus, A. terreus, A. niger and A. nidulans. In each case crude mycelial wall was extracted with an aqueous solution of Triton X-100 giving detergent-soluble material. Further fractionation was achieved by removing the detergent from this solution; the resultant precipitate was removed by centrifugation, and the aqueous supernatant was used as a source of soluble antigens. The sensitivity of these preparations was compared with that of water-soluble antigenic material, prepared from whole macerated mycelium, by double diffusion and counterimmunoelectrophoresis using homologous antisera and sera from patients suffering from aspergilloma and allergic bronchopulmonary aspergillosis. The selectivity of these antigenic preparations was monitored with heterologous antisera raised in rabbits. Batch variability was analysed for one strain of A. fumigatus using chemical and immunological methods. The nature of the antigenic sites involved in these reactions was investigated by studying the susceptibility of the preparations to proteolytic hydrolysis, periodate oxidation and concanavalin A treatment. The total protein and carbohydrate content of each fraction was determined and the constituent sugars analysed in an attempt to correlate chemical composition with antigenic activity.

Characterization of antigens from Aspergillus fumigatus. III. Comparison of antigenic relationships of clinically important aspergilli.

Antigenic relationships between strains of Aspergillus fumigatus, Aspergillus fumigatus var. elipticus, Aspergillus phialiseptus, Aspergillus flavus, and Aspergillus niger were analyzed by fused rocket immunoelectrophoresis and by skin tests. Seventy-three to 89 % of the numbers of antigens detected between strains and species of the A. fumigatus series were shared. The degree of sharing between antigens of A. flavus, A. fumigatus series, and A. niger was much lower and ranged from 19 to 35 %. In reciprocal skin tests in sensitized guinea pigs, similar relationships were shown. Three fractions of A. fumigatus extract proved to be markedly specific for this species. Cross reactivity was associated primarily with carbohydrate and glycoprotein fractions.

Analysis of Aspergillus nidulans conidial antigens and their prevalence in other Aspergillus species.

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. These elements constitute the main vehicle for the transmission of this and other pathogenic Aspergillus species and are the starting point of the different forms of aspergillosis. In order to use A. nidulans as a potential source of useful antigens for the immunodiagnosis of these diseases, we have examined the total protein composition of conidial extracts of this fungus by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in gels of different percent T. Injection of SDS-extracted conidial proteins into rabbits allowed us to raise a battery of polyclonal antibodies which have defined some important immunogenic polypeptides. Several of these immunogens were both present in mycelial extracts and recognized by antimycelium antibodies. Four of them, designated cdA, cdB, cdC, and cdE, were also found in conidial extracts of other pathogenic Aspergillus species. Only cdE was undetectable in cell extracts of the nonrelated species Fusarium culmorum and Phycomyces blakesleeanus.

Hemolytic, cytotoxic and complement inactivating properties of extracts of different species of Aspergillus.

Some of the biological properties of saline extracts of the mycelia of several species of the Aspergillus genus, namely, A. fumigatus, A. flavus, A. niger, A. nidulans, A. parasitucus and A. glaucus, were studied. Only the extract prepared from A. fumigatus was found to be hemolytic for sheep red blood cells. In contrast, all the extracts with the only exception of that of A. glaucus, had cytotoxic effects on Vero cells. Both, the hemolytic and cytotoxic constituents of the extracts were removable by adsorption with activated carbon. Heating of the extracts at 100 degrees C for 30 minutes also resulted in detoxification. In vivo studies, performed only with detoxified extracts of A. fumigatus, showed these were capable of depleting complement levels in guinea pigs. Complement inactivation was also found to occur in vitro and was cuased by all the extracts tested. Also triggered by the extracts was the conversion of serum C3 but not of purified C3, indicating that other serum factors are essential in the process. Despite the similarity in this respect with cobra venom factor, differences in activity after heating-negative in cobra venom factor-indicate that the complement inactivating substance/s present in the Aspergillus extracts differ from those of the snake product.