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Efficacy of cell-free antigens in evaluating cell immunity and inducing protection in a murine model of histoplasmosis.

Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a wide spectrum of disease when mycelial fragments are inhaled. Resistance to H. capsulatum is dependent on cellular immunity mediated by T cells and macrophages. Here we standardized the production of extracts containing cell-free antigens (CFAgs) and observed their efficacy in evaluating cellular immunity during murine histoplasmosis. CFAgs induced a more potent delayed-type hypersensitivity (DTH) response in H. capsulatum-infected mice than did histoplasmin-a classical antigen. This DTH response to CFAgs is able to determine the immune status of infected mice and to predict their death. Moreover, CFAgs stimulated spleen cells from immune mice to produce higher amounts of gamma interferon (IFN-gamma) in vitro. Finally, immunization with CFAgs protected against a lethal inoculum of H. capsulatum. These results demonstrate that CFAgs may be useful for the evaluation of cellular immune response and as a potential source for the development of a vaccine against histoplasmosis.

Animals↗

Risk of maxillary fungus ball in patients with endodontic treatment on maxillary teeth: a case-control study.

BACKGROUND: Maxillary sinus fungus ball (FB) is a noninvasive fungal disease commonly associated with symptoms of recurrent maxillary rhinosinusitis and/or extrusion of root canal filling material into the sinus. Chronic periapical inflammatory processes of dental origin are believed to be the risk factors in the pathogenesis of FB. The aim of this study was to determine whether endodontic treatment performed on maxillary molar, premolar, and canine teeth was a risk factor in the development of FB. METHODS: We designed a case-control study in which the cases were patients with FB admitted to the Department of Otorhinolaryngology at the University of Brescia between January 1990 and April 2005. For each case, 3 age-matched controls were randomly selected from the admission registry of the University Dental Clinic. Orthopantomography was used to detect endodontically treated maxillary molar, premolar, and canine teeth in both patients and controls. RESULTS: Of 102 patients with FB who were admitted during the study period, 91 (89.2%) had had endodontic treatment compared with 113 (36.9%) of 306 controls (chi square = 83.6601, P = 0.000; OR 14.13; 95% CI 7.25-27.54). The mean number (standard deviation; interquartile range [IQR]) of endodontic procedures was 1.39 (0.86; 1-2) in patients and 0.53 (0.81; 0-1) in controls (Mann-Whitney U test = -9.138, P = 0.0000). The interval between the endodontic treatment and the diagnosis of FB was available for 37 (36.3%) patients, and the median was 4 years (IQR 2-10). INTERPRETATION: Endodontic treatment on maxillary teeth is a strong risk factor for FB of the maxillary sinus.

Adult↗

The vasorelaxation of Antrodia camphorata mycelia: involvement of endothelial Ca(2+)-NO-cGMP pathway.

Antrodia camphorata, a medicinal fungus, has been used to treat cardiovascular diseases such as hypertension for many years. The purpose of this study was to examine the effects of mycelia extracts, from five Antrodia camphorata strains, on vascular tension and underlying mechanisms were explored. In isolated rat aortic rings, accession B86 caused concentration-dependent vasorelaxation with maximal relaxation of 40.34 +/- 7.53% whereas accessions 35398, 35396 and B71 had mild vasorelaxing effects. Strain B85 evoked potent vasorelaxation, partly through an endothelium-dependent mechanism that was inhibited by Nomega-nitro-L-arginine and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) but not by antagonist of K+ channels, tetraethylammonium. In cultured endothelial cells, B85 stimulated nitric oxide (NO) release and augmented the level of the intracellular Ca2+ concentration. HPLC and LC-MS-MS analysis revealed the presence of adenosine. Our results suggest that B85 produced strongest vasorelaxation in aortic preparations among five test strains. B85 acts in part on endothelial cells by activating the Ca(2+)-NO-cGMP pathway to reduce smooth muscle tone. However, K+ channels had no apparent roles. Adenosine could possibly be involved in the endothelium-dependent pathway of B85-induced vasorelaxation.

Adenosine↗

Purification of a major allergen, Asp f 2 binding to IgE in allergic bronchopulmonary aspergillosis, from culture filtrate of Aspergillus fumigatus.

BACKGROUND: Most cases of allergic bronchopulmonary aspergillosis (ABPA) are caused by the fungus Aspergillus fumigatus. Successful treatment of this disease depends on early diagnosis with the use of well-characterized and relevant antigens/allergens of the organism. OBJECTIVE: The aim of this study was to purify and characterize relevant proteins from A. fumigatus that could be used in the reliable diagnosis of ABPA. METHODS: Monoclonal antibodies were raised against A. fumigatus culture filtrate antigens. A Concanavalin A nonbinding protein fraction was purified with use of one of the monoclonal antibody immunoaffinity columns. The purified protein was analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis gel and Western blots. The sensitivity and specificity of the purified protein were evaluated by RAST and ELISA with sera from 25 patients with ABPA, from 10 with allergic asthma, and from 10 normal control subjects. RESULTS: The 37 kD Concanavalin A nonbinding protein reacted specifically with IgE antibodies in patients with ABPA. Among the 25 patients with ABPA studied, 96% had IgE antibody against the allergen, whereas none of the subjects with allergic asthma who had positive results on the skin prick test or normal control subjects had a reaction. Both RAST and ELISA results exhibited strong correlation with IgE binding. This allergen exhibited N-terminal sequence identity to a recombinant allergen Asp f 2. CONCLUSIONS: A 37 kD protein with complete N-terminal homology to Asp f 2 is a major allergen of A. fumigatus that significantly reacts with IgE antibody in patients with ABPA, but does not elicit reaction in Aspergillus-sensitive subjects with asthma and normal control subjects.

Allergens↗

Cryogenic X-ray crystal structure analysis for the complex of scytalone dehydratase of a rice blast fungus and its tight-binding inhibitor, carpropamid: the structural basis of tight-binding inhibition.

Scytalone dehydratase is a member of the group of enzymes involved in fungal melanin biosynthesis in a phytopathogenic fungus, Pyricularia oryzae, which causes rice blast disease. Carpropamid [(1RS,3SR)-2, 2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropa necarboxamide] is a tight-binding inhibitor of the enzyme. To clarify the structural basis for tight-binding inhibition, the crystal structure of the enzyme complexed with carpropamid was analyzed using diffraction data collected at 100 K. The structural model was refined to a crystallographic R-factor of 0.180 against reflections up to a resolution of 2.1 A. Carpropamid was bound in a hydrophobic cavity of the enzyme. Three types of interactions appeared to contribute to the binding. (i) A hydrogen bond was formed between a chloride atom in the dichloromethylethylcyclopropane ring of carpropamid and Asn-131 of the enzyme. (ii) The (chlorophenyl)ethyl group of carpropamid built strong contacts with Val-75, and this group further formed a cluster of aromatic rings together with four aromatic residues in the enzyme (Tyr-50, Phe-53, Phe-158, and Phe-162). (iii) Two hydration water molecules bound to the carboxamide group of carpropamid, and they were further hydrogen-bonded to Tyr-30, Tyr-50, His-85, and His-110. As a result of interactions between carpropamid and the phenylalanine residues (Phe-158 and Phe-162) in the C-terminal region of the enzyme, the C-terminal region completely covered the inhibitor, ensuring its localization in the cavity.

Amides↗

Characterization of cell wall degrading enzymes of Thanatephorus cucumeris.

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.

Cell Wall↗

Correlation between gliotoxin production and virulence of Aspergillus fumigatus in Galleria mellonella.

Aspergillus fumigatus is a pathogenic fungus capable of causing both allergic lung disease and invasive aspergillosis, a serious, life-threatening condition in neutropenic patients. Aspergilli express an array of mycotoxins and enzymes which may facilitate fungal colonisation of host tissue. In this study we investigated the possibility of using the insect, Galleria mellonella, for in vivo pathogenicity testing of Aspergillus species. Four clinical isolates of Aspergillus fumigatus and a single strain of Aspergillus niger were characterised for catalase and elastase activity and for the production of gliotoxin. Gliotoxin is an immunosuppressive agent previously implicated in assisting tissue penetration. Results illustrated a strain dependent difference in elastase activity but no significant difference in catalase activity. Gliotoxin production was detected in vitro and in vivo by Reversed Phase-High Performance Liquid Chromatography, with highest amounts being produced by A. fumigatus ATCC 26933 (350 ng/mg hyphae). Survival probability plots (Kaplan-Meier) of experimental groups infected with Aspergillus conidia indicate that G. mellonella is more susceptible to fungal infection by A. fumigatus ATCC 26933, implicating a critical role for gliotoxin production rather than growth rate or enzymatic activity in the virulence of A. fumigatus in this model.

Animals↗

Cryptococcal necrotizing fasciitis with multiple sites of involvement in the lower extremities.

BACKGROUND: Cryptococcal necrotizing fasciitis that is localized to the lower extremities is very rare. OBJECTIVE: We describe a case of a renal transplant recipient who presented with necrotizing fasciitis of the legs caused by Cryptococcus neoformans, a fungus that is rarely associated with this disease. METHODS: This is a case report with literature review. RESULTS: The patient was hospitalized, and the site of infection was debrided to the level of the periosteum. Cultures and histopathologic examination of biopsy material revealed an invasive deep-seated infection with a fungal organism that was consistent with C. neoformans. After 21 days on parenteral amphotericin B (Ambisome; Er-Kim Pharmaceuticals) treatment, the patient was switched to oral itraconazole (Itraspor; Janssen-Cilag Pharmaceuticals) 200 mg/day. He was discharged after 30 days of hospitalization with his wounds completely healed. He continued on oral fluconazole for a total course of 6 weeks. CONCLUSION: Systemic fungal infections continue to be an important cause of morbidity and mortality in transplant recipients. The insidious nature and atypical manifestations of these infections often delay diagnosis and therapy. In immunosuppressed patients, persistent fever that does not respond to antibacterial therapy should alert the physician to the possibility of fungal infection.

Amphotericin B↗

Topology, subcellular localization, and sequence diversity of the Mlo family in plants.

Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors.

Amino Acids↗

Comparison of immunomodulator mRNA and protein expression in the lungs of Stachybotrys chartarum spore-exposed mice.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. It can be separated into macrocyclic trichothecene-producing and nonproducing chemotypes based on secondary metabolite production. However, effects of spores of the two chemotypes on lung inflammatory responses are poorly understood. In this study, real-time reverse-transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to investigate time-course (1, 3, 6, 24, and 48 h post-instillation [PI]) relationships in mice intratracheally exposed to 300 spores/g body weight of a macrocyclic trichothecene-producing (JS 58-17) and a nonproducing (JS 58-06) S. chartarum isolate and of Cladosporium cladosporioides. There were marked differences in the magnitude and temporal patterns of mouse lung immune responses to intratracheal exposure to spores of these species at this spore dose. Both macrophage inflammatory protein 2 (MIP-2) and surfactant protein-D (SP-D) mRNA expression were significantly upregulated in lungs of JS 58-17-treated animals compared to that of all other treatment animals at 6 and 24 h PI. Heightened mRNA expression of these immunomodulators combined with comparatively depressed MIP-2 and tumor necrosis factor (TNF)-a protein expression suggests that the action of macrocyclic trichothecenes sequestered in 58-17 spores is involved. Interestingly, TNF-a protein expression in all spore treatment animal groups was also significantly increased over that in saline controls. Similarities in expression among all spore treatment animals suggest that chemicals other than toxic secondary metabolites, and possibly spore-sequestered 1,3-beta-D-glucan, may contribute to lung pathogenesis.

Animals↗

Maintenance of sex-linked deleterious alleles by selfing and group selection in metapopulations of the phytopathogenic fungus Microbotryum violaceum.

Microbotryum violaceum is a fungus that causes the sterilizing anther smut disease in many Caryophyllaceae. Its diploid teliospores are heterozygous at the mating-type locus, normally producing equal proportions of haploid sporidia of the two mating types. However, natural populations contain high frequencies of individuals producing sporidia of only one mating type. This mating-type ratio bias is caused by the presence of deleterious alleles at haploid phase ("haplo-lethals") linked to the mating-type locus. These haplo-lethals can be transmitted if there is conjugation among the products of meiosis (intratetrad selfing). Haplo-lethals still suffer from selective disadvantages, through reducing the infection probability of strains that carry them, and thus cannot persist in a panmictic population. We develop a realistic model of a metapopulation of M. violaceum on its host Silene latifolia. Simulations show that if intratetrad selfing rate is high, haplo-lethals can be maintained under a metapopulation structure because of founder effects and selection at the population level. Populations founded only by strains carrying haplo-lethals experience a lower extinction rate precisely because of their lower infection ability; they spread more slowly and sterilize fewer plants, thereby allowing their host population to grow more rapidly and therefore to be less prone to extinction.

Alleles↗

Comparison of inflammatory and cytotoxic lung responses in mice after intratracheal exposure to spores of two different Stachybotrys chartarum strains.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. While it can be separated into macrocyclic trichothecene- and atranone-producing chemotypes based on secondary metabolite production, effects of spores of the two chemotypes on lungs are poorly understood. In this study we used bronchoalveolar lavage fluid (BALF) to investigate dose-response (30, 300, 3000 spores/g body weight [BW]) and time-course (3, 6, 24, 48, 96 h post instillation [PI]) relationships in mice to exposure of macrocyclic trichothecene- (JS 58-17) and atranone-producing (JS 58-06) S. chartarum strains, as well as Cladosporium cladosporioides spores. BALF total protein, albumin, pro-inflammatory cytokine (IL-1beta, IL-6, and tumor necrosis factor-alpha [TNF-alpha]), and lactate dehydrogenase (LDH) concentrations showed significant (p < 0.05) fungal species (S. chartarum vs. C. cladosporioides) and strain (58-17 vs. 58-06), spore dose and time dependent changes. The no adverse effect level (NOAEL) due to exposure to spores of JS 58-17 and JS 58-06 was < 30 spores/g BW; for C. cladosporioides it was < 300 spores/g BW. At moderate and high S. chartarum doses, BALF composition reflects differences in strain toxicity while at the lowest dose, BALF composition of either S. chartarum strain were similar. This suggests that at low spore doses, it is spore sequestered factors common to both strains not strain dependent toxins that are contributing to lung disease onset.

Albumins↗

Purification, cloning and characterization of two xylanases from Magnaporthe grisea, the rice blast fungus.

Magnaporthe grisea, the fungal pathogen that causes rice blast disease, secretes two endo-beta-1,4-D-xylanases (E. C. 3.2.1.8) when grown on rice cell walls as the only carbon source. One of the xylanases, XYN33, is a 33-kD protein on sodium dodecyl sulfate-polyacrylamide gel and accounts for approximately 70% of the endoxylanase activity in the culture filtrate. The second xylanase, XYN22, is a 22-kD protein and accounts for approximately 30% of the xylanase activity. The two proteins were purified, cloned, and sequenced. XYN33 and XYN22 are both basic proteins with calculated isoelectric points of 9.95 and 9.71, respectively. The amino acid sequences of XYN33 and XYN22 are not homologous, but they are similar, respectively, to family F and family G xylanases from other microorganisms. The genes encoding XYN33 and XYN22, designated XYN33 and XYN22, are single-copy in the haploid genome of M. grisea and are expressed when M. grisea is grown on rice cell walls or on oatspelt xylan, but not when grown on sucrose.

Amino Acid Sequence↗

Chronic pulmonary histoplasmosis.

Chronic pulmonary histoplasmosis is best regarded as an opportunist or saprophytic infection of abnormal pulmonary spaces by a fungus of very low human pathogenicity. Tissue disease results from host immune response to dispersions of soluble antigen from these focal sources. There are two distinct types of clinical and radiological response. One is an acute or subacute illness manifested by often large segmental pneumonic lesions which tend to heal and are designated as early lesions. The other, usually developing as a complication of the first, is a chronic disease marked by persistent cavitation, low gard chronic illness, and a tendency to promote pulmonary fibrosis and often progressive pulmonary insufficiency. The early lesion is a segmental interstitial pneumonitis with central areas of infarct-like necrosis often adjacent to bullous disease and often outlining prominent emphysematous spaces which appear as radiolucencies. These radiological findings are further characterized by early clearing of the interstitial components, infarct-like contraction of the necrotic zones, obliteration of much of the contained emphysematous and bullous spaces, and healing attended by considerable loss of lung volume. Symptoms are variable but tend to be mild. Malaise, fatigability, low-grade fever, aching chest pain and mild cough lasting a few days to a few weeks are usual. Symptoms are ameliorated by rest. Rest and diminished activity are recommended as treatment. Under these circumstances, 80% of early lesions heal completely and probably most of these would heal spontaneously. Any subsequent course of the disease depends on whether or nor large air spaces, adjacent to or contained within the area of pneumonitis, become infected and persist as cavities. This occurs in 20% of early lesions. Once established, an infected cavity tends to persist and to be attended by symptoms of chronic bronchitis with chronic cough and sputum, fatigability, anorexia, and weight loss. Persisting thickwalled cavities often induce gradual development of pulmonary fibrosis, particulary in the lung bases, apparently from aspiration of antigenic material. This and the accelerated obstructive bronchopulmonary disease often lead to progressive pulmonary insufficiency. The use of amphotericin B is recommended for all persistent thick-walled cavities and in some circumstances surgical resection may be indicated.

Adolescent↗

The induction of sexual development and virulence in the smut fungus Ustilago maydis depends on Crk1, a novel MAPK protein.

MAP kinases (mitogen-activated protein kinases) are activated by dual phosphorylation on specific threonine and specific tyrosine residues that are separated by a single residue, and the TXY activation motif is a hallmark of MAP kinases. In the fungus Ustilago maydis, which causes corn smut disease, the Crk1 protein, a kinase previously described to have roles in morphogenesis, carries a TXY motif that aligns with the TXY of MAP kinases. In this work, we demonstrate that Crk1 is activated through a mechanism that requires the phosphorylation of this motif. Our data show that Fuz7, a MAPK kinase involved in mating and pathogenesis in U. maydis, is required to activate Crk1, most likely through phosphorylation of the TXY motif. Consistently, we found that Crk1 is also required for mating and virulence. We investigated the reasons for sterility and avirulence of crk1-deficient cells, and we found that Crk1 is required for transcription of prf1, a central regulator of mating and pathogenicity in U. maydis. Crk1 belongs to a wide conserved protein group, whose members have not been previously defined as MAP kinases, although they carry TXY motifs. On the basis of our data, we propose that all of these proteins constitute a new family of MAP kinases.

Amino Acid Motifs↗

Serinol phosphate as an intermediate in serinol formation in sugarcane.

A novel compound, serinol phosphate, was identified in sugarcane (Saccharum officinarum) clone 51NG97. It was produced by an enzyme-mediated transamination of dihydroxyacetone phosphate with either alanine, glutamate, aspartate, or glutamine serving equally well as an amino donor. Some detectable phosphatase activity was present in crude leaf enzyme preparation that hydrolyzed serinol phosphate. A proposal for a pathway of the biosynthesis of serinol in sugarcane was formulated.Serinol can serve as an "activator" of toxin production in attenuated cultures of the sugarcane pathogen Helminthosporium sacchari and it is present in susceptible clone 51NG97. Resistant clone H50-7209 does not possess serinol and likewise no dihydroxyacetone phosphate transaminase activity could be demonstrated in enzyme preparations of this clone. The concept of toxin activation in attenuated fungus cultures is briefly discussed relative to disease resistance and susceptibility.

Journal Article↗

Common sex-linked deleterious alleles in a plant parasitic fungus alter infection success but show no pleiotropic advantage.

Microbotryum violaceum is a fungus that causes the sterilizing anther smut disease in Caryophyllaceae. Its diploid teliospores normally produce equal proportions of haploid sporidia of its two mating types. However natural populations contain high frequencies of individuals producing sporidia of only one mating type ('biased strains'). This mating type-ratio bias is caused by deleterious alleles at haploid phase ('haplo-lethals') linked to the mating type locus that can be transmitted only by intra-tetrad selfing. We used experimental inoculations to test some of the hypotheses proposed to explain the maintenance of haplo-lethals. We found a disadvantage of biased strains in infection ability and high intra-tetrad mating rates. Biased strains had no higher competitive ability nor shorter latency and their higher spore production per flower appeared insufficient to compensate their disadvantages. These findings were only consistent with the hypothesis that haplo-lethals are maintained under a metapopulation structure because of high intra-tetrad selfing rates, founder effects and selection at the population level.

Basidiomycota↗

Production and characterization of two monoclonal antibodies specific for Plasmopara halstedii.

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.

Animals↗