Structural, mechanistic and functional insight into gliotoxin bis -thiomethylation in Aspergillus fumigatus

Abstract

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis—in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident.

Systems impact of zinc chelation by the epipolythiodioxopiperazine dithiol gliotoxin inAspergillus fumigatus: a new direction in natural product functionality

“…To our knowledge, this is the first report of an in vivo interaction between gliotoxin biosynthesis, dysregulated DTG presence and growth inhibition due to potential interference with Zn 2+ -associated growth systems. Moreover, it contributes to explaining the cryptic observation of Dolan et al that A. fumigatus DgliT::DgtmA is the most gliotoxinsensitive mutant observed to date, 15 and also why there are two distinct enzymatic activities which can prevent intracellular DTG accumulation. Overall, these data indicate the inhibitory potential of this endogenous, and potent, Zn 2+ chelator in A. fumigatus in particular, and possibly microorganisms in general.…”

“…9,13,14 Methylation of dithiol holomycin has been proposed as a back-up plan for self-protection, and Dolan et al revealed that A. fumigatus DgliT::DgtmA is significantly more sensitive to exogenous gliotoxin than a gliT-deficient mutant. 15 This suggests that the combined absence of both self-protection and negative regulation of DTG biosynthesis (or its intracellular presence) results in potent growth retardation -the precise cause of which is unknown. 10,15 Gliotoxin, other ETPs and dithiolopyrrolones have been shown to inhibit the activity of many enzymes, and functionality of specific proteins.…”

“…15 This suggests that the combined absence of both self-protection and negative regulation of DTG biosynthesis (or its intracellular presence) results in potent growth retardation -the precise cause of which is unknown. 10,15 Gliotoxin, other ETPs and dithiolopyrrolones have been shown to inhibit the activity of many enzymes, and functionality of specific proteins. [16][17][18][19][20][21] Several studies have demonstrated that the disulphide moiety of gliotoxin is responsible for most of the associated bioactivity of this molecule, whereas the S-methylated BmGT molecule is relatively inactive.…”

Functional Investigation of Iron-Responsive Microsomal Proteins, including MirC, in Aspergillus fumigatus

“…Plates were incubated statically at 37°C for 48 h. Mycelia were visualized by both differential interference microscopy and fluorescence microscopy using an Olympus Fluo View 1,000 Laser Scanning microscope, as per Dolan et al (2017). …”

Involvement of Sulfur in the Biosynthesis of Essential Metabolites in Pathogenic Fungi of Animals, Particularly Aspergillus spp.: Molecular and Therapeutic Implications

“…Indeed, we contend that the discovery of EGT may necessitate reconsideration of previous studies pertaining to the oxidative stress sensitivity of A. fumigatus, and possibly other fungi, following deletion of reactive oxygen species (ROS)-dissipating systems (Lessing et al, 2007;Lambou et al, 2010). We then move to reveal and illustrate that both gliotoxin and EGT biosynthesis (Sheridan et al, 2016;Dolan et al, 2017) has led to molecular rewiring in A. fumigatus, whereby Cys, GSH and SAM have become substrates for the biosynthesis of the aforementioned metabolites, along with playing a role in gliotoxin 'activation' and attenuation, leading to unexpected consequences for fungal metabolic systems (Owens et al, 2015). Finally, we introduce the concept of a 'cystathionine switch' in A. fumigatus which may play a role in fluxing thiol-containing metabolites toward different fates, depending on the prevailing cellular redox homeostatic requirements (Sheridan et al, 2016).…”