AGS3, an alpha(1-3)glucan synthase gene family member of Aspergillus fumigatus, modulates mycelium growth in the lung of experimentally infected mice.

The cell wall of human fungal pathogen Aspergillus fumigatus protects the fungus against threats from environment and interacts with the host immune system. Alpha(1-3)glucan is the major polysaccharide of Aspergillus fumigatus cell wall, and it has been shown to contribute to the virulence of diverse fungal pathogens. In A. fumigatus, three putative alpha(1-3)glucan synthase genes AGS1, AGS2 and AGS3 have been identified. AGS1 is responsible for cell wall alpha(1-3)glucan biosynthesis, but strains with deletions of either AGS1 or AGS2 are not defective in virulence [Beauvais, A., Maubon, D., Park, S., Morelle, W., Tanguy, M., Huerre, M., Perlin, D.S., Latgé, J. P., 2005. Two alpha(1-3) glucan synthases with different functions in Aspergillus fumigatus. Appl. Environ. Microbiol. 71, 1531-1538]. In contrast, we present evidence that AGS3 is also responsible for cell wall alpha(1-3)glucan biosynthesis and can modulate the virulence of A. fumigatus. An AGS3 deletion strain was found to produce faster and more robust disease than the parental strain in an experimental mouse model of aspergillosis. The apparent hyper-virulence in the AGS3-deleted mutant was correlated with an increased melanin content of the conidial cell wall, a better resistance to reactive oxygen species and a quicker germination rate. These results suggest an indirect role for AGS3 in virulence through an adaptive mechanism.

Specific molecular features in the organization and biosynthesis of the cell wall of Aspergillus fumigatus.

The cell wall of Aspergillus fumigatus is composed of a branched beta1,3 glucan covalently bound to chitin, beta1,3, beta1,4 glucans, and galactomannan, that is embedded in an amorphous cement composed of alpha1,3 glucan, galactomannan and polygalactosamin. The mycelial cell wall of A. fumigatus is very different from the yeast Saccharomyces cerevisiae cell wall, and in particular lacks beta1,6 glucans and proteins covalently bound to cell wall polysaccharides. The differences in cell wall composition between the mould A. fumigatus and the yeast S. cerevisiae are also reflected at the genomic level where unique features have been identified in A. fumigatus. A single gene codes for the glucan synthase catalytic subumit; this finding has lead to the development of a RNAi methodology for the disruption of essential genes in A. fumigatus. In contrast to the glucan synthase, multiple genes have been found in the chitin synthase and the alpha glucan synthase families; in spite of homologous sequences, each gene in each family have very different function. Similarly homologous mannosyltransferase genes are found in yeast and moulds but they lead to the synthesis of very different N-mannan structures. This chemo-genomic comparative analysis has also suggested that GPI-anchored proteins do not have a role of linker in the three dimensional organization of the fungal cell wall.

Two alpha(1-3) glucan synthases with different functions in Aspergillus fumigatus.

Alpha(1-3) glucan is a main component of the Aspergillus fumigatus cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in A. fumigatus, AGS1 and AGS2, are highly homologous to the AGS genes of Schizosaccharomyces pombe, which encode putative alpha(1-3) glucan synthases. The predicted Ags proteins of A. fumigatus have an estimated molecular mass of 270 kDa. AGS1 and AGS2 were disrupted in A. fumigatus. Both Deltaags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Deltaags1 presented a reduction in the alpha(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of A. fumigatus mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Deltaags1 and Deltaags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall alpha(1-3) glucan does not play a significant role in A. fumigatus pathogenicity.

A new experimental murine aspergillosis model to identify strains of Aspergillus fumigatus with reduced virulence.

Experimental animals are an obligate screen to investigate microorganism pathogenicity. Numerous animal models have been used to analyse the virulence of the opportunistic human pathogen Aspergillus fumigatus but none of the experimental models used previously have been satisfactory. This report discuss these models and presents a murine model of pulmonary aspergillosis that is very easy and the most adapted to compare the pathogenicity of A. fumigatus strains. Strains to be tested are inoculated intranasally and synchronously to mice and strains isolated from the lung of mice killed by the infection are typed. The number of colonies recovered is directly correlated to the virulence of the strain.

Membrane and cell wall targets in Aspergillus fumigatus.

Antifungal drugs directed against the human opportunistic fungal pathogen Aspergillus fumigatus are limited in number and ergosterol-targeted: the polyenes bind to the membrane ergosterol and the azoles block the ergosterol biosynthesis pathway. The efficacy of the drugs currently available for clinical use (amphotericin B and itraconazole) is limited and the frequent occurrence of therapeutic failures in the treatment of invasive aspergillosis emphasizes the need for the development of new agents. Cell wall biosynthetic pathways have been recognized for a long time as essential and unique specific drug targets. Recent studies of the chemical organization of the cell wall of A. fumigatus together with comparative analysis of yeast cell wall data have shown that beta 1-3 glucan branching and chitin-beta 1-3 glucan binding are essential exocellular enzymatic steps in cell wall biosynthesis. The enzymes involved in the biosynthesis and remodeling of cell wall polysaccharides especially in A. fumigatus are reviewed.

The first description of rheumatoid arthritis. Unabridged text of the doctoral dissertation presented in 1800.

This unabridged version of the dissertation presented in 1800 by Augustin Jacob Landré-Beauvais for his medical doctorate describes a disease different in many ways from the condition known since Hippocrates as gout. The patients were nine long-term residents of the Salpêtrière hospice in Paris. After reviewing the main features of ordinary or regular gout, Landré-Beauvais points out that the disease he calls "asthenic gout" exhibits several distinctive features, including predominance in women, a chronic course, involvement of many joints from the onset, and a decline in general health. Despite the tentative tone of his title ("Should one recognize...), he clearly is convinced that he is describing an as yet unreported entity, as indicated by the last sentence of his dissertation: "...we must recognize the existence of a new form of gout under the designation primary asthenic gout" And although he stops short of emancipation from the term "gout", which had been used for centuries to designate specific joint manifestations, he makes several keenly discerning observations -- particularly regarding the influence of psychological factors, the need for gentle treatments, and the inappropriateness of bloodletting - thus breaking free from contemporary doctrine. This text is acknowledged as the first description of rheumatoid arthritis. A brief biography of Landré-Beauvais is provided.

Glucan synthase complex of Aspergillus fumigatus.

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.

Molecular mechanisms of virulence in fungus-host interactions for Aspergillus fumigatus and Candida albicans.

Research on fungi that cause opportunistic infections has increased dramatically during the past few years, largely because these organisms cause significant morbidity and mortality. Most of this research has focused on defining the virulence factors produced by these pathogens, as well as developing methods for the diagnosis of fungal diseases. With regard to studies on the biology of Candida albicans, it is now possible to isolate genes, disrupt their expression, and observe the specific effects of gene disruption on virulence and growth of the organism. Moreover, growth and virulence of this pathogen is also being studied and the effect of environmental factors on gene expression investigated. This subject is especially important in view of the fact that C. albicans can colonize and invade a number of sites in the human body. Thus, its ability to grown in the oral and vaginal tracts, as well as in blood, requires the organism to adapt to a variety of environmental stresses. Here we present observations on the growth, morphogenesis and virulence of the opportunistic fungi C. albicans and Aspergillus fumigatus.

[Detection of chromosome anomalies using in situ hybridization on fetal tissue sections]. / Détection d'anomalies chromosomiques par hybridation in situ sur coupes de tissu foetal.

The use of in situ hybridization to detect and characterize chromosome anomalies on cytogenetic preparations is now largely applied in clinical laboratories. Its use in embryofetopathology on formalin-fixed and paraffin-embedded tissues, when an aneuploid condition is suspected but a routine chromosomal analysis is not possible was assessed in the present study. Control values of hybridization signals obtained after different enzymatic digestion protocols have been established on normal fetal tissues. Tissue conservation and fetal age as well as other parameters have also been analysed. A successful hybridization has been achieved in most cases. Failure of hybridization was observed in specimens associated with extensive tissular lysis. In conclusion, the application of in situ hybridization on fetal tissues is very useful to detect chromosome anomalies in embryofetopathology.

Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans.

A dipeptidyl-peptidase IV was purified from the culture medium of the human-pathogenic fungus Aspergillus fumigatus. The enzyme has an apparent molecular mass of 95 kDa and contained approximately 10 kDa of N-linked carbohydrate. This glycoprotein is antigenic and has all characteristics of the class IV dipeptidyl-peptidases: removal of Xaa-Pro and to a lesser extent Xaa-Ala dipeptides from the N termini of peptides, including bioactive peptides such as neuropeptide Y, [des-Arg1] bradykinin, and glucagon-like peptide 1, activity at neutral pH, and presence in the amino acid sequence of the Gly-X-Ser-X-Gly consensus motif of the serine-hydrolases and the putative catalytic triad (Ser613, Asp690, His725) of the dipeptidyl-peptidases. Moreover, the last 200 amino acids displayed 60 to 65% similarity with the other dipeptidyl-peptidases IV from rat, mouse, human, and yeast. However, unlike the other dipeptidyl-peptidases, the dipeptidyl-peptidase IV of A. fumigatus is a secreted enzyme with a cleavable signal peptide. Expression of a recombinant dipeptidyl-peptidase IV of A. fumigatus has been attained in the yeast Pichia pastoris.

Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus.

A novel dipeptidyl-peptidase (DPP V) was purified from the culture medium of Aspergillus fumigatus. This is the first report of a secreted dipeptidyl-peptidase. The enzyme had a molecular mass of 88 kDa and contained approximately 9 kDa of N-linked carbohydrate. The expression and secretion of dipeptidyl-peptidase varied with the growth conditions; maximal intra- and extracellular levels were detected when the culture medium contained only proteins or protein hydrolysates in the absence of sugars. The gene of DPP V was cloned and showed significant sequence homology to other eukaryotic dipeptidyl-peptidase genes. Unlike the other dipeptidyl-peptidases, which are all intracellular, DPP V contained a signal peptide. Like the genes of other dipeptidyl-peptidases, that of DPP V displayed the consensus sequences of the catalytic site of the nonclassical serine proteases. The biochemical properties of native and recombinant DPP V obtained in Pichia pastoris were unique and were characterized by a substrate specificity limited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum. In addition, we showed that DPP V is identical to one of the two major antigens used for the diagnosis of aspergillosis.

Purification and characterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus.

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.

Unsaturated fatty acids are the active molecules of a glucan-synthase-inhibitory fraction isolated from entomophthoralean protoplasts.

A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, glucan synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of glucan synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 degrees C and were shown to be non-competitive and non-specific inhibitors of glucan synthase.

Cloning of two chitin synthase gene fragments from a protoplastic entomophthorale.

Chitin synthase expression was studied in spontaneously produced protoplasts and in hyphal bodies of the Entomophthorale species Entomophaga aulicae. The absence of wall in protoplasts was correlated to an absence of chitin synthase. Two chitin synthase activities with different biochemical characteristics have been detected in the hyphal bodies. Two chitin synthase gene fragments EaCHS1 and EaCHS2 of 600 bp were obtained using PCR amplification of genomic DNA. Their amino acid sequences showed 75% identity. Compared with other fungal chitin synthases, they belong to class II. EaCHS1 and EaCHS2 were used to probe total RNA from E. aulicae hyphal bodies and protoplasts. A single transcript of 2.4 kb hybridized only with EaCHS1 in protoplasts and hyphal bodies.

Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment.

The molecules that mediate cell-matrix recognition, such as fibronectins (FN) and integrins, modulate cell behavior. We have previously demonstrated that FN and the beta 1-integrins are used during neural crest cell (NCC) migration in vitro as well as in vivo, and that the FN cell-binding domains I and II exhibit functional specificity in controlling either NCC attachment, spreading, or motility in vitro. In the present study, we have analyzed the effect of changes in the integrin expression patterns on migratory cell behavior in vivo. We have generated, after stable transfection, S180 cells expressing different levels of alpha 4 beta 1 or alpha 5 beta 1 integrins, two integrins that recognize distinct FN cell-binding domains. Murine S180 cells were chosen because they behave similarly to NCC after they are grafted into the NCC embryonic pathways in the chicken embryo. Thus, they provide a model system with which to investigate the mechanisms controlling in vitro and in vivo migratory cell behavior. We have observed that either the overexpression of alpha 5 beta 1 integrin or the induction of alpha 4 beta 1 expression in transfected S180 cells enhances their motility on FN in vitro. These genetically modified S180 cells also exhibit different migratory properties when grafted into the early trunk NCC migratory pathways. We observe that alpha 5 and low alpha 4 expressors migrate in both the ventral and dorsolateral paths simultaneously, in contrast to the parental S180 cells or the host NCC, which are delayed by 24 h in their invasion of the dorsolateral path. Moreover, the alpha 4 expressors exhibit different migratory properties according to their level of alpha 4 expression at the cell surface. Cells of the low alpha 4 expressor line invade both the ventral and dorsolateral pathways. In contrast, the high expressors remain as an aggregate at the graft site, possibly the result of alpha 4 beta 1-dependent homotypic aggregation. Thus, changes in the repertoire of FN-specific integrins enable the S180 cells to exploit different pathways in the embryo and regulate the speed with which they disperse in vivo and in culture. Our studies correlate well with known changes in integrin expression during neural crest morphogenesis and strongly suggest that neural crest cells that migrate into the dorsolateral path, i.e., melanoblasts, do so only after they have upregulated the expression of FN receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the 1,3-beta-glucan synthase of Aspergillus fumigatus.

1,3-beta-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors--GTP, NaF, sucrose and EDTA--added during the extraction procedure, were essential for optimal 1,3-beta-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (Km for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.

Warts among workers in poultry slaughterhouses.

The objective of the study was to identify the prevalence of warts in poultry slaughterhouses. A questionnaire was administered to 1,194 workers, of whom 569 completed it. The prevalence of warts (Papilloma virus) was found to be 28.5% in slaughterhouses. In the general population it is 7-10%, reaching a peak in individuals around the age of 14 a. In the poultry slaughterhouses, the prevalence was highest (38.7%) in the age category 25-29 a, and among those who had been working 4 to 6 a (40.8%). The factors present in the work environment showed a significant correlation (alpha less than 0.05) with the presence of warts were the following: steel-mesh gloves that were too large for the wearer, work with a saw, handling of cold objects, and high humidity levels. It was concluded that mild abrasion of the skin (too-large gloves) and high humidity facilitate cutaneous infection by the virus, whereas the handling of cold objects and work with a saw provoke local vasoconstriction which could hinder the immune reaction.