Identification of Cryptic Products of the Gliotoxin Gene Cluster Using NMR-Based Comparative Metabolomics and a Model for Gliotoxin Biosynthesis

Forseth, Ry R.; Fox, Ellen M.; Chung, Dawoon; Howlett, Barbara J.; Keller, Nancy P.; Schroeder, Frank C.

doi:10.1021/ja2029987

Cited by 85 publications

(92 citation statements)

References 32 publications

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“…These metabolites did not contain free thiols, and as noted above they did not possess growth-inhibitory properties associated with organic extracts of wild-type organic extracts. Also, the observed m/z did not correspond to any metabolite present in the A. fumigatus wild-type intracellular metabolome (13) or secretome of the A. fumigatus ⌬gliG strain (11,31). Moreover, this mass (393 to 395 Da) is greater than that of gliotoxin, 326 Da, and strongly suggests the presence of either a shunt metabolite(s) or biosynthetic intermediate of higher mass than gliotoxin, as previously observed (31).…”

Section: Discussionsupporting

confidence: 52%

“…Interestingly, this work also demonstrated that gliP and gliI deletion mutant metabolomes did not contain any gliZ-dependent compounds. While this might be expected for GliP absence, which normally catalyzes the initiation step in gliotoxin biosynthesis (1), it is unclear why gliI (a putative aminotransferase) deletion resulted in the complete absence of gliZ-dependent metabolites, although the absence of gliZ and gliP expression in A. fumigatus ⌬gliI has been noted (13).…”

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confidence: 99%

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The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

et al. 2012

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The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H 2 O 2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P ‫؍‬ 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H 2 O 2 (P < 0.01), unexpectedly, exogenous gliotoxin relieved H 2 O 2 -induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ⌬gliK 26933 deletion mutant. The A. fumigatus ⌬gliK 26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ⌬gliK 26933 mutant than in those of the wild type (P ‫؍‬ 0.0024 [fold difference, 24] and P ‫؍‬ 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ⌬gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ⌬gliK 26933 mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P ‫؍‬ 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ⌬gliK 46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus. Gliotoxin was discovered in 1936, and its structure was elucidated in 1943 (19, 38). Much initial interest in the molecule was directed toward exploiting its antifungal activities; however, after observations that gliotoxin exhibited growth-inhibitory effects against animal cells, it was posited as a putative anti-cancer or organ transplant rejection agent, and a large number of subsequent studies investigated the mechanism of action of gliotoxin against mammalian cell types (3, 24, 37).Until recently, the mechanism of gliotoxin biosynthesis had proven to be especially refractory to analysis, despite multiple studies between 1960 and 1985 involving radiolabeled precursor feeding and analysis of proteins produced during gliotoxin biosynthesis (2, 18). Since the discovery of the gliotoxin gene cluster gli (14), now known to contain 13 genes, the function of a number of cluster genes has been established by a combination of targeted gene deletion and phenotypic and biochemical analyses (4, 10, 11, 13, 22, 31-33, 34, 36). gliZ [a Zn(II) 2 -Cys(6) binuclear cluster domain transcription factor] deletio...

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Section: Discussionsupporting

confidence: 52%

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confidence: 99%

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

et al. 2012

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“…11 Two partial structures (units a and b, Figure 1) were assigned by interpretation of the 2D NMR data (Table 1). In unit a, the connectivity of the three olefinic protons (H-7, H-8, and H-9) was unambiguously assigned from the COSY data.…”

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Gliotoxin Analogues from a Marine-Derived Fungus, Penicillium sp., and Their Cytotoxic and Histone Methyltransferase Inhibitory Activities

et al. 2011

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Seven gliotoxin-related compounds were isolated from the fungus Penicillium sp. strain JMF034, obtained from deep sea sediments of Suruga Bay, Japan. These included two new metabolites, bis(dethio)-10a-methylthio-3a-deoxy-3,3a-didehydrogliotoxin (1) and 6-deoxy-5a,6-didehydrogliotoxin (2), and five known metabolites (3−7). The structures of the new compounds were elucidated by analysis of spectroscopic data and the application of the modified Mosher's analysis. All of the compounds exhibited cytotoxic activity, whereas compounds containing a disulfide bond showed potent inhibitory activity against histone methyltransferase (HMT) G9a. None of them inhibited HMT SET7/9. A fter intensive investigation of secondary metabolites of marine macroorganisms for almost half a century, 1 some marine natural product chemists concluded that new structural entities from these organisms were nearly exhausted. Therefore, they turned their attention to marine microorganisms as an untapped source of secondary metabolites. 2,3 With this trend in mind we have been searching for cytotoxic metabolites produced by marine-derived fungi. We found that a Penicillium sp. (strain JMF034) isolated from deep-sea sediments collected in Suruga Bay exhibited potent cytotoxic activity. From the culture medium we isolated a series of metabolites related to gliotoxin 4 and gliotoxin itself as the active constituents, among which two metabolites are new. Gliotoxin is a representative member of the epipolythiodioxopiperazine (ETP) class of fungal metabolites.5 Due to its potent cytotoxicity toward cancer cell lines, this compound has been considered as a lead for anticancer agents.5 Recently, dimeric ETPs were shown to inhibit histone methyltransferase (HMT). 6 These observations prompted us to examine the HMT-inhibitory activity of the metabolites, some of which showed significant activity. Here we describe structure elucidation of the new compounds.The culture medium of Penicillium sp. JMF034 was extracted with EtOAc, and the combined EtOAc layers were fractionated by solvent partitioning, gel permeation, and reversed-phase column chromatographies followed by ODS-HPLC to yield compounds 1−7. The known compounds were identified as bis(dethio)bis(methylthio)gliotoxin (3), 7 bis(dethio)bis-(methylthio)-5a,6-didehydrogliotoxin (4), 8 5a,6-didehydrogliotoxin (5), 9 gliotoxin (6), 10 and gliotoxin G (7), 8 on the basis of ESIMS and 1 H NMR data.The molecular formula of compound 1 was determined to be C 14 H 16 N 2 O 3 S on the basis of the HRESIMS data. Analysis of the 1 H NMR spectrum (Table 1) in conjunction with the HSQC data revealed two singlet methyls, a pair of nonequivalent methylene protons, two oxygen-or nitrogensubstituted methines, five olefinic protons, two of which were a pair of exomethylene protons, and an exchangeable proton. Even though the number of protons was small, interpretation of the COSY spectrum was complex due to the overlap of H-5a and H-6, the absence of coupling between H-6 and H-7, and the observation of long-ran...

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“…Indeed, existing paradigms of gliotoxin ( Fig. 1) as a toxin and the perspective of the disulfide bridge-containing (oxidized) form as the final, or only, product are undergoing significant reconsideration (6)(7)(8)(9)(10)(11).…”

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“…Moreover, multiple S-methylated intermediates have been identified in wild-type A. fumigatus but not in an A. fumigatus strain deficient in gliotoxin biosynthesis (⌬gliZ) (9). Although bisdethiobis(methylthio)gliotoxin (BmGT) (Fig.…”

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Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus

et al. 2015

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Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ⌬gliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ⌬gliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ⌬gliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ⌬gliK strain, but not the ⌬gliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence. Biosynthesis, self-protection mechanisms, and functionality of gliotoxin and related epidithiodiketopiperazine (ETP) molecular species, such as chaetocin and acetylaranotin, are attracting ever-increasing attention as a consequence of findings from highthroughput genome sequencing projects, application of gene deletion technologies, and mass spectrometric analytical methodologies (1-5). Indeed, existing paradigms of gliotoxin (Fig. 1) as a toxin and the perspective of the disulfide bridge-containing (oxidized) form as the final, or only, product are undergoing significant reconsideration (6-11).Self-protection against disulfide-containing metabolites appears to be essential in both fungi and bacteria. It has been demonstrated that the gliotoxin oxidoreductase GliT (12), encoded within the gli cluster, protects Aspergillus fumigatus against exogenous gliotoxin and is essential for gliotoxin biosynthesis (12, 13). A similar mechanism for self-protection against holomycin in Streptomyces clavulige...

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Identification of Cryptic Products of the Gliotoxin Gene Cluster Using NMR-Based Comparative Metabolomics and a Model for Gliotoxin Biosynthesis

Cited by 85 publications

References 32 publications

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

Gliotoxin Analogues from a Marine-Derived Fungus, Penicillium sp., and Their Cytotoxic and Histone Methyltransferase Inhibitory Activities

Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus

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