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Aspergillus fumigatus has become a leading cause of fungal morbidity and mortality, especially in immunocompromised patients. This fungus is able to grow as a multicellular community and produce a hydrophobic extracellular matrix (ECM), mainly composed of galactomannan and α-1,3 glucans, to protect itself from host defenses and antimicrobial drugs. This matrix envelops the fungus hyphae, binding them into a contiguous sheath on the colony surface, forming a biofilm and increasing the fungal resistance to adverse environmental factors. Adherence to host cells and resistance to physical removal play a key role in fungal colonization and invasion of the host and in a wide range of infections. Here we show that, by using atomic force spectroscopy, it is possible to exploit the peculiar hydrophobicity of the biofilm components (i.e., cell walls, ECM) to detect the biofilm spread, its growth, and lysis on rough surfaces. By means of this approach, we demonstrate that alginate lyase, an enzyme known to reduce negatively charged alginate levels in microbial biofilms, reduces the biofilm adhesion forces suggesting a loss of ECM from the biofilm, which could be used to enhance pharmacological treatments.

Live-cell imaging techniques are now routinely used to study filamentous fungi. This has been very much facilitated by the development of a wide range of novel microscope technologies, new fluorescent probes (vital dyes and fluorescent proteins), and major advances in computing hardware and software. Here we show what can be achieved with imaging living cells of Aspergillus fumigatus and A. nidulans in vitro using confocal laser scanning microscopy. Basic techniques for successful live-cell imaging are described, and results are shown from imaging the dynamics of the growing colony margin, nuclear division, mitochondria, endoplasmic reticulum, Golgi and asexual structures. Other live-cell imaging studies that have been performed on Aspergillus are also summarized.

alpha-Glucosidase I regulates trimming of the terminal alpha-1,2-glucose residue in the N-glycan-processing pathway, which plays an important role in the quality control system in mammalian cells. However, the consequence of glucose trimming of the N-glycan in filamentous fungi is unclear. We identified the gene encoding alpha-glucosidase I in the human opportunistic fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene resulted in a defective N-glycan processing of the proteins secreted by A. fumigatus. Although the Afcwh41 was not essential for hyphal growth and virulence, a severe reduction in conidia formation and a temperature-sensitive deficiency of cell wall integrity (CWI) were observed. Also, abnormalities of polar growth and septation were observed during conidial germination and hyphal elongation of the mutant. Our results suggest that Afcwh41 was involved in CWI, polarity, septation, and conidiation in A. fumigatus, probably by affecting the proper function of the proteins that are required for cell wall synthesis.

This is a case of vocal cord aspergillosis in a 36-year-old woman whose chief complaint was progressive hoarseness and vocal fatigue of one month duration. These symptoms followed the use of systemic administration of penicillin, cefotaxime natrium and dexamethasone to treat her rhinitis and asthma. Fiberoptic laryngoscopy revealed whitish plaques involving both vocal cords. Microscopic examination of KOH preparations and histopathology studies of tissue revealed dichotomously branching, hyaline, septate hyphae. Morphological and molecular biological identification of the fungal growth in cultures inoculated with clinical specimens from the patient indicated that the etiologic agent was Aspergillus fumigatus. The patient was cured with oral itraconazole without any side effects.

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.