Secondary metabolite and mycotoxin from A. fumigatus

Aspergillus fumigatus, an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report thatA. fumigatusescapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] metabolism through Gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P3and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P3 Together, our findings identify phosphoinositide metabolism as a critical upstream target of Gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P3signaling during membrane ruffling and phagocytosis. [1]

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Gliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10min with 30-100ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin-induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia. [2]

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Aspergillus fumigatus is able to internalize into lung epithelial cells to escape from immune attack for further dissemination. We previously reported that Gliotoxin, a major mycotoxin of A. fumigatus, promotes this internalization; however, the mechanism remained unclear. Here, we report that gliotoxin is able to induce cofilin phosphorylation in A549 type II human pneumocytes. Either too high or too low a level of cofilin phosphorylation blocked the gliotoxin-induced actin cytoskeleton rearrangement and A. fumigatus internalization. LIM domain kinase 1 (LIMK1) and its upstream small GTPases (Cdc42 and RhoA, but not Rac1) predominantly mediated the gliotoxin-induced cofilin phosphorylation and A. fumigatus internalization. Simultaneously, gliotoxin significantly stimulated an increase in cAMP; however, adding an antagonist of PKA did not block gliotoxin-induced A. fumigatus internalization. In vivo, exogenous gliotoxin helped gliotoxin synthesis deficient strain gliPΔ invade into the lung tissue and the lung fungal burden increased markedly in immunosuppressed mice. In conclusion, these data revealed a novel role of gliotoxin in inducing cofilin phosphorylation mostly through the Cdc42/RhoA-LIMK1 signaling pathway to promote actin cytoskeleton rearrangement and internalization of A. fumigatus into type II human pneumocytes. [3]

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Chronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)- dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA-bound NOTCH2 complexes are less sensitive to the γ-secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum-derived secondary metabolite Gliotoxinas a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA-bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor κB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified Gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL [4].

Exogenous Gliotoxin Promotes A. fumigatus Invasion in Lung Tissues in Mice Immunosuppressed With Hydrocortisone Acetate [3]
In our previous study, gliotoxin was able to promote A. fumigatus internalization into lung epithelial cells at a low concentration of 50 ng/ml in vitro (Jia et al., 2014), which is much lower than 200 ng/ml used in previous studies (Stanzani et al., 2005; Kupfahl et al., 2006a, 2006b), it was interesting to test whether gliotoxin promotes A. fumigatus invasion into lung epithelium in vivo. Ten to twelve mice that were immunosuppressed with hydrocortisone acetate were infected with each strain of A. fumigatus (wild-type B5233, deletion of the peptide synthetase gliP type gliPΔ, or gliPΔ plus exogenous 50 ng/ml gliotoxin). The survival rate of the gliPΔ mutant infected mice was significantly higher than that of wild-type B5233 infected mice, and exogenous gliotoxin could reduce the survival of mice infected with the gliPΔ mutant with statistical significance (Figure 6A). The mice infected with wild-type B5233 or glipΔ mutant strain plus exogenous gliotoxin, but not those infected with the gliPΔ mutant strain showed a significant decline in body weight at 3 days post-infection (Figure 6B). Furthermore, exogenous addition of gliotoxin significantly enhanced the pulmonary fungal burden of mice (Figures 6C,D). Pathological staining showed a stronger inflammatory response and more severe invasion of hyphae (black arrow) in the lung tissue, causing tissue necrosis, in mice infected with wild-type B5233 than in the other two mouse groups. In lung tissues of mice infected with gliPΔ alone, the fungus was mainly located in the bronchia, accompanied with an inflammatory response in the peripheral lung tissue. In comparison, infection with gliPΔ plus exogenous gliotoxin caused stronger inflammation and a large number of bronchial epithelial shedding, and in some instances even invasion into the lung tissue (Figure 6E). These results suggested that exogenous gliotoxin promoted A. fumigatus invasion in lung tissues in the mouse model of invasive aspergillosis immunosuppressed with hydrocortisone acetate in the initial phase of infection.

Gliotoxin and other pharmacological treatments.[1]
Gliotoxin was diluted in DMSO and utilized at a final concentration of 500 ng/ml (1.53 µM) in all instances, except for experiments in which the toxin was serially diluted. Gliotoxin was added while the cells were in serum-free medium and allowed to act for 30 min before further experimental manipulations were carried out. In the cases where gliotoxin challenge was followed by the addition of a second compound (e.g., phorbol myristate acetate [PMA]), gliotoxin was not removed from the medium. PMA and wortmannin were both used at 100 nM for the indicated periods. The adenylate cyclase activator forskolin and the competitive inhibitor of cAMP signaling (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) trimethylammonium (cAMPS-Rp) were utilized at final concentrations of 100 and 200 µM, respectively. Where indicated, cells were pretreated with cAMPS-Rp before being exposed to either gliotoxin or forskolin.

The mice were infected with A. fumigatus alone or with A. fumigatus mixed with exogenous 50 ng/ml Gliotoxin . For survival analysis, the mice were weighed every day after infection. In the majority of cases, the end-point for survival experimentation was death. At the relevant time-points post-infection, mice were sacrificed and lungs tissues were weighed, homogenized, and then serially diluted onto Sabouraud agar plates for incubation at 37°C. After 20 h, the number of colonies in lung tissues (number of CFU/g) was also counted and calculated. For histopathological examination, the lung tissue sections obtained from mice from each group at 72 h post-infection were dissected, fixed in 10% (vol/vol) formaldehyde, and stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). Images were taken using a BX51 microscope with an Olympus DP71 camera using brightfield illumination at 200× or 400× magnification.

[1]. Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis. MBio. 2016 Apr 5;7(2):e02242.

[2]. Gliotoxin from Aspergillus fumigatus affects phagocytosis and the organization of the actin cytoskeleton by distinct signalling pathways in human neutrophils. Microbes Infect. 2007 Jan;9(1):47-54. Epub 2006 Dec 12.

[3]. Gliotoxin Induces Cofilin Phosphorylation to Promote Actin Cytoskeleton Dynamics and Internalization of Aspergillus fumigatus Into Type II Human Pneumocyte Cells. Front Microbiol. 2019 Jun 18;10:1345.

[4]. Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells. Br J Haematol. 2013 Mar;160(5):618-29.

Gliotoxin is a pyrazinoindole with a disulfide bridge spanning a dioxo-substituted pyrazine ring; mycotoxin produced by several species of fungi. It has a role as a mycotoxin, an immunosuppressive agent, an EC 2.5.1.58 (protein farnesyltransferase) inhibitor, a proteasome inhibitor and an antifungal agent. It is an organic disulfide, a pyrazinoindole, an organic heterotetracyclic compound and a dipeptide.
Gliotoxin has been reported in Trichoderma virens, Trichoderma deliquescens, and other organisms with data available.
Gliotoxin is a sulfur-containing antibiotic produced by several species of fungi, some of which are pathogens of humans such as Aspergillus, and also by species of Trichoderma, and Penicillium. Gliotoxin possesses immunosuppressive properties as it may suppress and cause apoptosis in certain types of cells of the immune system, including neutrophils, eosinophils, granulocytes, macrophages, and thymocytes. (L1941)
A fungal toxin produced by various species of Trichoderma, Gladiocladium fimbriatum, Aspergillus fumigatus, and Penicillium. It is used as an immunosuppressive agent.

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Macrophages and neutrophils thus seem to carry out independent yet complementary tasks in shaping the course of Aspergillus pathobiology: the former are responsible for the quick and efficient phagocytosis of inhaled conidia, while the latter may play a more active role in contending with complex and extensive hyphal networks, which are simply too large for macrophages to phagocytose. By subverting phosphoinositide signaling—and therefore interfering with both actin cytoskeletal dynamics and integrin function—gliotoxin may impair the function of both kinds of professional phagocytes, thereby ensuring the survival of its progeny while keeping hyphal networks clear from neutrophil onslaught. [1]

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In conclusion, the present study elucidated for the first time that gliotoxin, an essential toxin from A. fumigatus, stimulates cofilin phosphorylation through the Cdc42/RhoA-LIMK1 and cAMP/PKA-LIMK1 signaling pathways, and consequently, modulates actin cytoskeleton rearrangement to facilitate A. fumigatus internalization into lung epithelial cells (Figure 7). [3]

C13H14N2O4S2

326.38

326.039

C, 47.84; H, 4.32; N, 8.58; O, 19.61; S, 19.65

67-99-2

6223

White to off-white solid powder

1.8±0.1 g/cm3

699.7±55.0 °C at 760 mmHg

153.5ºC

377.0±31.5 °C

0.0±5.0 mmHg at 25°C

1.814

0.52

2

6

1

21

621

4

CN1C(=O)[C@]23CC4=CC=C[C@@H]([C@H]4N2C(=O)[C@]1(SS3)CO)O

FIVPIPIDMRVLAY-RBJBARPLSA-N

InChI=1S/C13H14N2O4S2/c1-14-10(18)12-5-7-3-2-4-8(17)9(7)15(12)11(19)13(14,6-16)21-20-12/h2-4,8-9,16-17H,5-6H2,1H3/t8-,9-,12+,13+/m0/s1

(3R,5aS,6S,10aR)-6-hydroxy-3-(hydroxymethyl)-2-methyl-2,3,5a,6-tetrahydro-10H-3,10a-epidithiopyrazino[1,2-a]indole-1,4-dione

Gliotoxin; gliotoxin; 67-99-2; Aspergillin; CCRIS 4025; Gliotoxin from Gliocladium fimbriatum; NSC 102866; UNII-5L648PH06K; BRN 0050675; Aspergillin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMF : 5 mg/mL (~15.32 mM)
DMSO : ~5 mg/mL (~15.32 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)