Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low‐dose mouse infection model

Abstract

SummaryGliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the nonribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wildtype strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

“…While this approach is acceptable, the number of fungal metabolites which can be present in organic extracts, combined with the possibility of peptide generation due to unwanted proteolysis, demands a more rigorous confirmation of gliotoxin presence. LC-MS analysis of gliotoxin fulfils this criterion; however, this is a relatively specialised technique and is not available to all researchers [12,15,16]. Moreover, alternative bioassay formats have also been proposed for detection of gliotoxin and related epipolythiodioxopiperazines, of fungal origin, at levels of 18-20 ng well −1 [19,25].…”

“…Studies on gliotoxin biosynthesis, which have demonstrated the role of the non-ribosomal peptide synthetase, GliP, in mediating the initial step of gliotoxin formation, have also deployed LC-MS to detect the presence and absence of gliotoxin in wild-type and mutant (ΔgliP) cultures from A. fumigatus [16]. Moreover, using a chemical reduction and alkylation strategy, thiol and disulphide absence has been confirmed in a gliotoxin-like metabolite isolated from an A. fumigatus mutant deficient in gliotoxin biosynthesis [17].…”

“…Moreover, using a chemical reduction and alkylation strategy, thiol and disulphide absence has been confirmed in a gliotoxin-like metabolite isolated from an A. fumigatus mutant deficient in gliotoxin biosynthesis [17]. To date, matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-ToF MS) has not been used for the detection of gliotoxin, as matrix components can interfere with the detection of low molecular mass metabolites and gliotoxin fragments during ionisation, thereby rendering detection impossible [16,18].…”

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis

“…However, it is unclear whether these secondary metabolites contribute to virulence, because the role and production of these compounds during infection has hardly been investigated yet. An exception is the immune modulator gliotoxin, which was re-isolated from infected mouse lungs Kupfahl et al, 2006), but, also for this metabolite, the impact on virulence is still under discussion. A hint for the general impact of secondary metabolites on virulence derives from investigations on A. fumigatus mutants, which carry a deleted leaA gene.…”

Parallels in Fungal Pathogenesis on Plant and Animal Hosts

“…This latter assumption has been tested recently by two research groups who disrupted the peptide synthetase gene in the biosynthetic pathway and studied the effect of these non-gliotoxin-producing mutants in vitro and in vivo in animal models. The mutants showed reduced cytotoxic activity on cells such as mast cells or macrophages but did not affect the survival of mice with invasive aspergillosis (23,47). Thus, gliotoxin does not appear to be required for the pathogenicity of the fungus in immunocompromised mice.…”