c.i.-fluorescent-brightening-agent-28 and Aspergillosis

If the mycelium of Aspergillus fumigatus is very short-lived in the laboratory, conidia can survive for years. This survival capacity and extreme resistance to environmental insults is a major biological characteristic of this fungal species. Moreover, conidia, which easily reach the host alveola, are the infective propagules. Earlier studies have shown the role of some molecules of the outer conidial layer in protecting the fungus against the host defense. The outer layer of the conidial cell wall, directly in contact with the host cells, consists of α-(1,3)-glucan, melanin, and proteinaceous rodlets. This study is focused on the global importance of this outer layer. Single and multiple mutants without one to three major components of the outer layer were constructed and studied. The results showed that the absence of the target molecules resulting from multiple gene deletions led to unexpected phenotypes without any logical additivity. Unexpected compensatory cell wall surface modifications were indeed observed, such as the synthesis of the mycelial virulence factor galactosaminogalactan, the increase in chitin and glycoprotein concentration or particular changes in permeability. However, sensitivity of the multiple mutants to killing by phagocytic host cells confirmed the major importance of melanin in protecting conidia.

Topics: Aspergillosis; Aspergillus fumigatus; Azoles; Benzenesulfonates; Caspofungin; Cell Wall; Chitin; Congo Red; Fungal Proteins; Glucans; Glycoproteins; Humans; Hydrophobic and Hydrophilic Interactions; Melanins; Monocytes; Mycelium; Phagocytes; Polysaccharides; Pyocyanine; Spores, Fungal; Virulence Factors

Invasive fungal diseases (IFD) are an important cause of morbidity and mortality in immunocompromised patients, and early diagnosis and management are a challenge. We evaluated the clinical utility of computed tomography (CT)-guided percutaneous lung biopsies in diagnosing IFD.. Between 2003 and 2014, we analyzed 2671 CT-guided lung biopsies, from which 157 were IFD associated; we aimed to determine microbiological-based diagnostic accuracy of calcofluor white staining (CFWS), culture, Aspergillus antigen detection (GM), broad-range fungal PCR, and Aspergillus PCR per sample.. 127 (81%) specimens were microscopically positive for any fungal elements, 30 (19%) negative. Aspergillus and non-Aspergillus like hyphae were obtained in 85 (67%) and 42 (33%) specimens, respectively. CFWS positivity was defined as proof of infection. Sensitivity, specificity, and positive (PPV) and negative predictive (NPV) values for CT scan were 100, 44, 80, and 100%, for Aspergillus PCR 89, 58, 88, and 58%, for broad-range fungal PCR 90, 83, 95, and 90%, and for GM 94, 83, 95, and 90%. The most common CT features were patchy opacifications with central necrosis (78%) or cavern defects (50%), less common were air bronchograms (39%) or ground glass halos (39%), and all other features were rare. The overall pneumothorax rate subsequent to biopsy was 19%, but in only 2% of all cases the placement of a chest tube was indicated. One case of fatal air embolism occurred.. CT-guided lung biopsies have high diagnostic accuracy in terms of microscopic examination, and complication rates are low. Molecular-based and antigen tests applied on fungal hyphae-positive specimens showed comparable results.

Topics: Aged; Antigens, Fungal; Aspergillosis; Aspergillus; Austria; Benzenesulfonates; Biopsy; Female; Humans; Immunocompromised Host; Lung; Lung Diseases, Fungal; Male; Middle Aged; Polymerase Chain Reaction; Specimen Handling; Tomography, X-Ray Computed

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.

Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Benzenesulfonates; beta-Glucans; Caspofungin; Cell Wall; Chitin; Echinocandins; Galactose; Glucosyltransferases; Hyphae; Lipopeptides; Mannans; Mutation; Phenotype; Polysaccharides

Mitogen-activated protein kinase (MAPK) signaling pathways are involved in the regulation of various cellular responses in eukaryotes. In fungal pathogens they are of special interest because of their possible contribution to pathogenicity. Bioinformatic analysis of the genome of the most prevalent airborne human pathogenic fungus Aspergillus fumigatus, revealed the presence of four distinct MAPK-encoding genes. Here, we present the detailed functional analysis of one of these MAPKs, MpkA. Comparative analysis revealed similarities of MpkA with MAPKs involved in cell wall integrity signaling of other fungi. Accordingly, the analysis of mpkA deletion mutants revealed severe sensitivity of the mutants against cell wall active compounds, drastical alterations of the fungal morphology and increased resistance against oxidative stress. The expression of mpkA was induced by cell wall damaging conditions. Despite its involvement in cell wall signaling no influence on virulence of the deletion of mpkA was observed in a murine infection model.

Topics: Animals; Antifungal Agents; Artificial Gene Fusion; Aspergillosis; Aspergillus fumigatus; Benzenesulfonates; beta-Galactosidase; Caffeine; Cell Wall; Congo Red; Diamide; Gene Deletion; Gene Expression Regulation, Fungal; Genes, Reporter; Genetic Complementation Test; Hydrogen Peroxide; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Oxidants; Oxidative Stress; Signal Transduction; Sodium Dodecyl Sulfate; Survival Analysis; Virulence; Vitamin K 3

We assessed Calcofluor white staining, Aspergillus polymerase chain reaction, and a galactomannan enzyme immunoassay for diagnosis of fungal infection with use of computed tomography-guided percutaneous lung biopsy specimens obtained from 61 patients. The sensitivity and specificity of computerized tomography, Aspergillus polymerase chain reaction, and galactomannan enzyme immunoassay were 100% and 50%, 100% and 86%, and 88% and 94%, respectively.

Topics: Adult; Aspergillosis; Aspergillus; Benzenesulfonates; Biopsy; Contrast Media; False Positive Reactions; Female; Galactose; Humans; Immunocompromised Host; Immunoenzyme Techniques; Lung Diseases, Fungal; Male; Mannans; Middle Aged; Polymerase Chain Reaction; Prospective Studies; Sensitivity and Specificity; Tomography, X-Ray Computed

The pathogenic mould Aspergillus fumigatus can cause severe infections in immunocompromised patients. Phagocytosis of inhaled conidia is an early and crucial event in the defense of A. fumigatus infections. Here we describe a novel antibody-independent assay for quantification of phagocytosis, that in this study has been applied to different Aspergillus species, but that is in principle suitable for many fungi.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Benzenesulfonates; Biotin; Cell Line; Cytochalasin D; Fluorescent Dyes; Humans; Macrophages, Alveolar; Mice; Microscopy, Fluorescence; Phagocytosis

Aspergillus fumigatus is an opportunistic pathogenic fungus that predominantly infects the respiratory system. Penetration of the lung alveolar epithelium is a key step in the infectious process. The cytoskeleton of alveolar epithelial cells forms the cellular basis for the formation of a physical barrier between the cells and their surroundings. This study focused on the distinct effects of A. fumigatus on the actin cytoskeleton of A549 lung pneumocytes. Of the 3 major classes of cytoskeletal fibers--actin microfilaments, microtubules, and intermediate filaments--only the actin cytoskeleton was found to undergo major structural changes in response to infection, including loss of actin stress fibers, formation of actin aggregates, disruption of focal adhesion sites, and cell blebbing. These changes could be specifically blocked in wild-type strains of A. fumigatus by the addition of antipain, a serine and cysteine protease inhibitor, and were not induced by an alkaline serine protease-deficient strain of A. fumigatus. Antipain also reduced, by approximately 50%, fungal-induced A549 cell detachment from the plates and reduction in viability. Our findings suggest that A. fumigatus breaches the alveolar epithelial cell barrier by secreting proteases that act together to disorganize the actin cytoskeleton and destroy cell attachment to the substrate by disrupting focal adhesions.

Topics: Actins; Antipain; Aspergillosis; Aspergillus fumigatus; Benzenesulfonates; Cell Adhesion; Cell Line, Tumor; Cytoskeleton; Endopeptidases; Formazans; Humans; Immunohistochemistry; Lung; Microscopy, Confocal; Microtubules; Protease Inhibitors; Vinculin