Detection of Gliotoxin in Experimental and Human Aspergillosis

Abstract

Gliotoxin was measured in the lungs (mean, 3,976 ؎ 1,662 ng/g of tissue) and sera (mean, 36.5 ؎ 30.28 ng/ml) of mice with experimentally induced invasive aspergillosis (IA), and levels decreased with antifungal therapy. Gliotoxin could also be detected in the sera of cancer patients with documented (proven or probable) IA.Aspergillus fumigatus, the saprophytic mold associated with the majority of cases of human invasive aspergillosis (IA), is known to produce a variety of secondary metabolites during invasive (hyphal) growth (12). One of the most abundantly produced metabolites is the epipolythiodioxopiperazine metabolite gliotoxin, which has a broad spectrum of immunosuppressive activity in animals and humans (15). Gliotoxin has been shown in vitro to inhibit activation of the transcription factor NF-B (9), neutrophil and macrophage oxidative killing (7,8,14), macrophage phagocytosis (7, 8), antigen-mediated lymphocyte stimulation and cytotoxic-T-cell activation (6, 15), and gamma interferon production by CD4 ϩ lymphocytes (17). At higher concentrations (Ͼ250 ng/ml), gliotoxin induces apoptosis in macrophages and lymphocytes by a mechanism distinct from its antiphagocytic effects (16). Theoretically, the release of gliotoxin by Aspergillus during growth in tissues could aid in the evasion by the fungus of innate and professional effector immune cells.(This work was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 14 to 17 September 2003, abstr. M-1015 and M-1243 We hypothesized that gliotoxin production is involved in the pathogenesis of IA and could be a marker of invasive infection with A. fumigatus. To this end, we used a liquid chromatographytandem mass spectrometry (LC-MS-MS) assay to measure gliotoxin concentrations in the lungs and sera of mice with experimentally induced invasive pulmonary aspergillosis (IPA). Additionally, we screened unselected banked serum samples for gliotoxin from a cohort of cancer patients with or without documented evidence of IA.Serum samples were collected under institutional review board protocol LAB 02-200 at The University of Texas M. D. Anderson Cancer Center (MDACC). All animal studies were performed under the guidance and institutional standards of The University of Texas MDACC and The University of Houston Institutional Care and Animal Use Committees.Serum or tissue samples were prepared for analysis by methanol extraction and precipitation of proteins followed by centrifugation at 10,000 ϫ g. LC-MS-MS in negative mode was run using pairs of ions at an m/z of 324.9/260.8 for gliotoxin and 137.9/107.9 for the internal standard 3-nitrophenol. Concentrations of gliotoxin were assessed by interpolation of unknown samples to a seven-point standard curve (0.25 to 25 ng/ml) for purified gliotoxin (Sigma, St. Louis, Mo.) prepared in mouse sera or uninfected mouse lung homogenate. Off-scale unknown samples were diluted 100-to 1,000-fold and reassayed against the standard curve. Mean inter-and intraassay coefficients ...

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

“…In addition, Lewis et al showed that gliotoxin was present in sera and lungs of mice suffering from experimentally induced invasive aspergillosis by LC-MS analysis [15]. These authors also demonstrated that gliotoxin could be detected, by LC-MS, in cancer patients with either proven or probable invasive aspergillosis [15].…”

“…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].…”

Functional convergence of gliP and aspf1 in Aspergillus fumigatus pathogenicity

“…Gliotoxin is necessary for maximal virulence in experimental mouse models of invasive pulmonary aspergillosis [16,17]. Furthermore, it is detectable in the serum of patients with invasive aspergillosis, suggesting that it also contributes to the pathogenesis of this disease in humans [18]. …”

Nonribosomal cyclic peptides: specific markers of fungal infections

“…48 This compound, which has a broad spectrum of immunosuppressive activities in animals and humans, 49 was recently determined in the lungs and sera of mice with experimentally induced aspergillosis. 50 It was also detected in the sera of human cancer patients with documented invasive aspergillosis. Tandem mass spectrometry in negative ion mode and selective reaction monitoring electro spray ionization (ESI) were used in this study.…”

“…These findings led the authors to speculate on the role of secreted metabolites in the pathogenesis and persistence of opportunistic infections. 50 An immunoassay detection clinically more acceptable than mass spectral approaches has therefore been developed: gliotoxin and helvolic acid were conjugated to thyroglobulin for antisera generation in rabbits as well as to bovine serum albumin (BSA). 51 The antiserum could then be subsequently used for the detection of both free and conjugated haptens.…”