Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin

Abstract

Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxin called restrictocin. To investigate the role of restrictocin in IPA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption. These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay. Western blot analysis of one disruptant, using an anti-restrictocin monoclonal antibody, confirmed that the toxin is not produced. The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media. The pathogenicity of two disruptants was assessed in a murine model of IPA. There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant. In addition, histological examination of infected lung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclear leucocyte response. Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.

Variation in virulence of Aspergillus fumigatus strains in a murine model of invasive pulmonary aspergillosis

“…Tang et al [6] and Smith et al [9] decreased the number of conidia to achieve 100% mortality (8 x 1 O5 conidia/mouse) by administration of the corticosteroid (1 12.5 mgkg) in five stages. In the present study, the inoculum required to produce infection was optimised to give reproducible survival curves and a 100% mortality rate.…”

“…However, the most recent data have suggested that these enzymes are not major virulence factors . Recently, other metabolites, including cytotoxins have been studied [9][10][11]. The results of DNA typing methods have suggested a genomic polymorphism between A. fumigatus strains [ 12-1 61.…”

Pathogenesis ofAspergillus fumigatusin Invasive Aspergillosis

“…The deletion of the res gene to create mutants deficient in the ribonucleotoxin restrictocin did not have an effect on virulence or fungal burden in neutropenic models of IA (185). However, culture filtrates of restrictocin mutants were unable to suppress neutrophil-mediated hyphal damage, in contrast to filtrates from wild-type strains (84).…”

Use of Recombinant Mitogillin for Improved Serodiagnosis ofAspergillus fumigatus-Associated Diseases

“…Mitogillin is a small basic protein of approximately 18 kDa with cytotoxic activity released by A. fumigatus (16,17,31). Together with the related toxins A. fumigatus allergen I (AspfI), restrictocin from Aspergillus restrictus (20), and ␣-sarcin from Aspergillus giganteus (26), mitogillin is a member of a family of conserved RNases that cleave a single phosphodiester bond of the 29S rRNA of eukaryotic ribosomes (11,16,17).…”