Experimental avian aspergilosis

One-day-old chicks were infected by aerosolized conidia ofAspergillus fumigatus orA. flavus. Plaques containing viable organisms were observed on the third day of infection. Although plaques persisted for several weeks, cultures could not be recovered from them after about ten days. Flourescent antibody methods revealed an immune response to both agents within three days of challenge. Precipitin titers againstAspergillus active protein (AAP) did not appear until the tenth day, and was not detected after seven weeks. Aspergillus polysaccharide (APS) failed to react antigenically in infected birds. AAP was separated by Sephadex chromatography into nine distinct fractions. One fraction from each species was associated with an immediate and one with a delayed type of skin response in wattles. A low molecular weight fraction which lacked skin reactivity was an effective antigen in precipitin tests. Except for a weak skin reaction toA. fumigatus APS in rabbits, skin tests with extracts and APP fractions correlated with wattle reactions in chicks.     

Chemical and immunological studies on mycobacterial polysaccharides. 1. Purification and properties of polysaccharides from human tubercle bacilli

Defatted human tubercle bacilli, Aoyama B strain, were extracted with 0.1 n NaOH for 24 hr, and the crude polysaccharide fraction was precipitated by the addition of 5 volumes of ethyl alcohol. A yield of 17.8 g of crude polysaccharides was obtained from 800 g of bacilli. The crude polysaccharide was further fractionated into seven fractions by fractional precipitation with ethyl alcohol. Each fraction was purified by successive chromatography on Dowex 50 and diethylaminoethyl cellulose, and by gel filtration on Sephadex G-75 and G-200. Optical rotation and gas chromatographic analyses of purified polysaccharide showed that these polysaccharides contained glucan mannan, arabinomannan, and arabinogalactan. Each polysaccharide was almost completely free from nitrogen, and no tuberculin reaction was produced by 100 mug of each material. Arabinomannan and arabinogalactan showed precipitin reaction, complement fixation, and passive hemagglutination reaction with rabbit antiserum against heat-killed whole bacilli (Aoyama B). In guinea pigs sensitized with Aoyama B bacilli, arabinomannan and arabinogalactan provoked anaphylactic shock when injected intravenously, and Arthus type reaction when injected intracutaneously. With the use of rabbit antiserum, arabinomannan and arabinogalactan showed passive anaphylactic shock, passive cutaneous anaphylaxis, and Prausnitz-Küstner type reactions in guinea pigs. By immunodiffusion analysis, it was shown that the antigenic determinant of arabinomannan was different from that of arabinogalactan.     

Characterization of immunologically active peptides from the cell wall of T.mentagrophytes

A low molecular weight fraction from chitinase digested cell walls ofT. mentagrophytes containing both polysaccharide and peptide moieties was found to have immunological reactivity at both the cellular and humoral level. This fraction (UM₂(a)) was further degraded by treatment with either a combination of pronase and carboxypeptidase A or with trypsin. Peptides were separated from the carbohydrate-rich fraction by ultrafiltration. The carbohydrate-rich fraction retained the ability to induce both immediate and delayed skin reactions in sensitized guinea pigs and to stimulate the proliferation of sensitized lymphocytesin vitro. The peptide moieties retained reactivity in that they caused delayed reactions and lymphocyte proliferation but were unable to induce immediate or Arthus reactions in sensitized animals. Tryptic peptides from UM₂(a) were purified by ion exchange chromatography. A high proportion of these peptides demonstrated immunological activity at both the cellular and humoral level since they were capable of inducing delayed reactions and/or lymphocyte transformation, as well as being capable of blocking the complement fixation reaction between UM₂ (a) and specific antiserum.This work was supported by grant number 6411 from the Medical Research Council of Canada.A. Kh. Al-Rammahy was supported by a scholarship from the Ministry of High Education, Iraq.     

The Skin-Reactive Antigens of Mycoplasma pneumoniae

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the organism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 μg. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform–methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Studies on Delayed Hypersensitivity of Antigens Isolated from Mycoplasma pneumoniae Cells

Cells of Mycoplasma pneumoniae Mac strain were fractionated into acetone-soluble and insoluble fractions. Acetone-insoluble fractions were digested with pronase and further purified by chromatography on Sephadex G-75, yielding three water-soluble fractions which were free from lipid and consisted mainly of polysaccharide-protein complex. All these water-soluble fractions possessed eliciting antigenicity to delayed hypersensitivity for M. pneumoniae as measured by skin reactions and macrophage migration inhibition tests, but not to complement-fixing antibodies. In contrast, the acetone-soluble fraction was reactive with the complement-fixing antibodies but not for the delayed hypersensitivity.     

Development of two inbred strains of rats and characteristics of their skin reactions.

Two inbred strains of rat (Donryu and Sprague-Dawley strains) were developed. The skin reactions of these strains immunized with M. tuberculosis, hen egg albumin (OVA) or hen egg lysozyme and challenged with the purified protein derivative (PPD) or each antigen were even and uniform. The Donryu strain showed a typical Arthus reaction with petechiae and edema and a negligible delayed skin reaction, whereas the Sprague-Dawley strain showed a poor Arthus reaction and a typical delayed skin reaction with central necrosis and induration. The Arthus reaction or delayed skin reaction could be passively transferred to recipient rats of each strain by immune sera or sensitized peritoneal exudate cells (PEC), respectively.     

Skin test-active substance prepared from culture filtrate of Fonsecaea pedrosoi

Ethanol-precipitated substance (EP) was prepared from culture filtrate of Fonsecaea pedrosoi. EP was separated into two components by passing through a Sephadex G-50 column; the faster passing component was referred to as EP-1, the slower as EP-2. EP-1 and EP-2 were evaluated as an antigen for detecting cutaneous delayed hypersensitivity in patients with chromomycosis. EP-1 elicited positive delayed skin reactions in all of 8 patients with chromomycosis, of which 7 caused by F. pedrosoi and one by Exophiala jeanselmei. Healthy subjects, patients with sporotrichosis and patients with tinea barbae failed to react to EP-1. These results indicate that EP-1 is a useful tool for detecting cutaneous delayed hypersensitivity in patients with chromomycosis caused by F. pedrosoi. It was found that precipitin test using EP-1 as an antigen had little diagnostic value in chromomycosis. EP-2 did not show antigenic activity in both skin and precipitin reactions.     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.     

Coccidioidin sensitivity in control,immunized and infected guinea pigs

Summary Delayed hypersensitivity to potent coccidioidin developed in guinea pigs immunized with deadC. immitis arthrospores and guinea pigs infected with an aerosol ofC. immitis arthrospores at weeks 1 and 2. Delayed hypersensitivity in control animals sensitized by repeated intradermal testing developed at weeks 3 and 4. The delayed hypersensitivity responses were characterized grossly by indurations larger than 25 mm² and could be seen at 6 and 24 hours after testing. Retesting reduced the size of the 24 hour indurations when compared to virginal reactions. The retest delayed reactions in infected animals had indurations at 24 and 48 hours that were larger than those in the other groups.In those animals that were skin test positive but not challenged no tube precipitins, agar gel precipitins, CF antibodies, anaphylaxis or immediate hypersensitivity were detected. Because of the inability to detect precipitins the early phase of the hypersensitivity seen at 6 hours was not considered an Arthus reaction.     

Grucuronic acid-containing glycopeptide from squid cartilage

A glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its K_av value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 μmol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37°C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.     

Study on Inhibition by Ribocitrin of Glucan Production of Streptococcus mutans E49

α-Glucan produced by crude dextransucrase (CEP) of Streptococcus mutans E49 was separated into the following three fractions: a water-insoluble glucan fraction (designated as IG), a water-soluble glucan fraction with a wide distribution of molecular weight (SG-1) and an oligosaccharide (SG-2). Formation of these products, which had characteristic courses, were remarkably reduced in the presence of ribocitrin. Production of IG and SG-1 by CEP and the inhibitory activity of ribocitrin were highly pH-dependent. With regard to dextran T10, ribocitrin inhibited IG production competitively.     

Aspergillus fumigatus devoid of cell wall β‐1,3‐glucan is viable,massively sheds galactomannan and is killed by septum formation inhibitors

Echinocandins inhibit β‐1,3‐glucan synthesis and are one of the few antimycotic drug classes effective against Aspergillus spp. In this study, we characterized the β‐1,3‐glucan synthase Fks1 of Aspergillus fumigatus, the putative target of echinocandins. Data obtained with a conditional mutant suggest that fks1 is not essential. In agreement, we successfully constructed a viable Δfks1 deletion mutant. Lack of Fks1 results in characteristic growth phenotypes similar to wild type treated with echinocandins and an increased susceptibility to calcofluor white and sodium dodecyl sulfate. In agreement with Fks1 being the only β‐1,3‐glucan synthase in A. fumigatus, the cell wall is devoid of β‐1,3‐glucan. This is accompanied by a compensatory increase of chitin and galactosaminogalactan and a significant decrease in cell wall galactomannan due to a massively enhanced galactomannan shedding. Our data furthermore suggest that inhibition of hyphal septation can overcome the limitations of echinocandin therapy. Compounds inhibiting septum formation boosted the antifungal activity of caspofungin. Thus, development of clinically applicable inhibitors of septum formation is a promising strategy to improve existing antifungal therapy.     

The skin-reactive antigens of Mycoplasma pneumoniae.

Guinea pigs experimentally infected with Mycoplasma pneumoniae or immunized with the orgnaism in combination with Freund's complete adjuvant developed a delayed hypersensitive skin reaction following on intradermal injection of the M. pneumoniae antigen. The amount of protein necessary to produce the delayed skin reaction was as low as 0.01 mug. When the sonicated whole cells were extracted with aqueous acetone, the delayed skin reactivity was found mostly in the acetone insoluble (lipid-depleted) fraction. On the other hand, the lipid fraction which was isolated by a chloroform-methanol extraction of the acetone-soluble fraction and had a high titer of complement-fixing activity, exhibited little delayed skin reactivity. The lipid-depleted antigens as the whole cell antigens produced delayed skin reactivities in human patients.     

Aspergillus fumigatus exoβ(1‐3)glucanases family GH55 are essential for conidial cell wall morphogenesis

The cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched β(1‐3)glucan, to which the chitin and the galactomannan are covalently bound. Softening of the cell wall is an essential event during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosyl hydrolases. In this study, we characterised the role of the glycosyl hydrolase GH55 members in A. fumigatus fungal morphogenesis. We showed that deletion of the six genes of the GH55 family stopped conidial cell wall maturation at the beginning of the development process, leading to abrogation of conidial separation: the shape of conidia became ovoid, and germination was delayed. In conclusion, the reorganisation and structuring of the conidial cell wall mediated by members of the GH55 family is essential for their maturation, normal dissemination, and germination.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.     

Serological studies on Aspergillus fumigatus

Summary 1) Extract was obtained by repeated cryolysis of acetone-dried mycelia ofAsp. fumigatus killed by formalin. High titer immune serum could be easily obtained by immunizing rabbits with this extract.2) FI antigen prepared from mycelial extract by alcoholic precipitation exhibited species-specificity when examined by the precipitin test.3) FII, prepared from FI by chloroform-butanol treatment, was no better than FI in antigenic activity.4) FI could be used as antigen in the skin test with rabbits infected withAsp. fumigatus, when the final reading was based on the redness (larger than 5 mm in diameter) 36 hours after the injection of antigen.     

Allergenicity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations in Mice and Guinea Pigs

Guinea pigs were injected subcutaneously with mycobacterial ribosomal fraction incorporated in Freund's incomplete adjuvant and tested 6 and 12 weeks later by the intradermal injection of 0.5 μg (25 TU) of Purified Protein Derivative. No evidence of delayed-type hypersensitivity could be detected in these animals, although large necrotic reactions were obtained in guinea pigs sensitized with living, attenuated mycobacterial cells. Mice also were vaccinated by the intraperitoneal injection of mycobacterial ribosomal fraction or ribonucleic acid (RNA) and tested for sensitivity to tuberculin at various subsequent times. No evidence of true tuberculin hypersensitivity could be detected at any time, although what appeared to be small Arthus type reactions were seen in mice given the largest vaccinating doses. Attempts to recall tuberculin sensitivity in vaccinated mice by the intravenous injection, 4 weeks after vaccination of living cells, of either the virulent or attenuated mycobacterial strains were unsuccessful. Instead, when the virulent cells were injected, a suppression of footpad reactivity was noted in animals made sensitive to tuberculin by the previous intraperitoneal injection of viable attenuated mycobacterial cells. Both guinea pigs and mice, vaccinated as described above, were also skin tested or footpad tested, respectively, with 2 μg of the ribosomal fraction or RNA used for vaccination. No evidence of true tuberculin hypersensitivity could be obtained; instead, in guinea pig skin very small dermonecrotic areas were noted, and in mice swelling and redness of the footpad occurred to an equal extent in both vaccinated and nonvaccinated mice. The possible role of tuberculin hypersensitivity in acquired immunity to tuberculosis is discussed, and the conclusion is reached that its part, if any, is minor.     

Glycoprotein biosynthesis by organ cultures of hamster trachea

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [¹⁴C]glucosamine and [³H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as “intracellular”, gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and “intracellular” material; it labelled extensively with both glucosamine and fucose and had a molecular weight of approx. 5000 (as judged by its elution from Sephadex G-75). This fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.     

Studies on glycopeptides derived from acidic glycoproteins of guinea-pig serum

1. Fraction I, a fraction containing acidic glycoproteins, isolated from guinea-pig serum, was digested with Pronase after removal of sialic acid and a major and a minor glycopeptide fraction were isolated by chromatography with Sephadex G-25 and G-50. 2. The major fraction was examined by various methods and shown to contain several glycopeptides. Estimates of molecular weight of the glycopeptide fractions were obtained. Although some variation appeared to occur, the glycopeptides were not grossly heterogeneous with respect to size. An average prosthetic group was estimated to contain about 15 sugar residues. 3. Aspartic acid was the principal amino acid present in the fractions and in all subfractions of the major fraction investigated. Where examined, ammonia was liberated on acid hydrolysis in approximately equimolar amounts to the aspartic acid present. The carbohydrate composition of the fractions was also determined. 4. The glycopeptides showed relatively little degradation in alkaline solution. 5. These results suggest that an N-acylglycosylamine bond involving aspartic acid forms the major type of linkage between carbohydrate and polypeptide. The isolation of a compound with the composition and chromatographic properties of 2-acetamido-1-(l-beta-aspartamido)-1,2-dideoxy-beta-d-glucose supports this view, and indicates that N-acetylglucosamine is the sugar involved in at least many linkages. 6. Fraction I contains some glycoproteins that are susceptible to Pronase and one or more others that resist digestion before the removal of sialic acid. A brief examination revealed some similarities between prosthetic groups derived from both kinds of glycoprotein.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.