Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

Characterization of a cell-wall acid phosphatase (PhoAp) in Aspergillus fumigatus

In the filamentous fungus Aspergillus fumigatus, the vast majority of the cell-wall-associated proteins are secreted proteins that are in transit in the cell wall. These proteins can be solubilized by detergents and reducing agents. Incubation of a SDS/beta-mercaptoethanol-treated cell-wall extract with various recombinant enzymes that hydrolyse cell-wall polysaccharides resulted in the release of a unique protein in minute amounts only after incubation of the cell wall in the presence of 1,3-beta-glucanase. Sequence analysis and biochemical studies showed that this glycoprotein, with an apparent molecular mass of 80 kDa, was an acid phosphatase (PhoAp) that was active on both phosphate monoesters and phosphate diesters. PhoAp is a glycosylphosphatidylinositol-anchored protein that was recovered in the culture filtrate and cell-wall fraction of A. fumigatus after cleavage of its anchor. It is also a phosphate-repressible acid phosphatase. The absence of PhoAp from a phosphate-rich medium was not associated with a reduction in fungal growth, indicating that this cell-wall-associated protein does not play a role in the morphogenesis of A. fumigatus

Filamentous fungi recovered from the water distribution system of a Belgian university hospital

A study was carried out over a 4-month winter period in order to assess the presence of filamentous fungi in the water distribution system of the University Hospital of Liège. A total of 197 hot and cold water samples were collected from the main water supply lines and from the taps at three different hospital sites. Overall, filamentous fungi were recovered from 55% and 50% of the main water distribution system and tap water samples, respectively, with a mean of 3.5 ± 1.5 colony forming units per 500 ml water. Nine different genera were identified, all belonging to the Hyphomycetes class. Aspergillus spp. were recovered from 6% of the samples of the water distribution system and A. fumigatus was the most frequently recovered species (66.6%). However, this species was not isolated from water taps. Fusarium spp. was predominant at one site, where it was found in 28% of tap water samples. No Aspergillus spp. but some Fusarium spp. isolates were identified in samples collected from high-risk units. Filters were introduced at the point-of-use in the haematology unit after completion of the study. The findings of the present study confirm the need for further documented studies to evaluate the safety of the hospital water system and to define new preventive measures