Downes, Shane G. and Owens, Rebecca A. and Walshe, Kieran and Fitzpatrick, David A. and Dorey, Amber and Jones, Gary W. and Doyle, Sean (2023) Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion depletion. Scientific Reports, 13 (1). ISSN 2045-2322

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Abstract

Overcoming antimicrobial resistance represents a formidable challenge and investigating bacterial growth inhibition by fungal metabolites may yield new strategies. Although the fungal non-ribosomal peptide gliotoxin (GT) is known to exhibit antibacterial activity, the mechanism(s) of action are unknown, although reduced gliotoxin (dithiol gliotoxin; DTG) is a zinc chelator. Furthermore, it has been demonstrated that GT synergises with vancomycin to inhibit growth of Staphylococcus aureus. Here we demonstrate, without precedent, that GT-mediated growth inhibition of both Gram positive and negative bacterial species is reversed by Zn2+ or Cu2+ addition. Both GT, and the known zinc chelator TPEN, mediate growth inhibition of Enterococcus faecalis which is reversed by zinc addition. Moreover, zinc also reverses the synergistic growth inhibition of E. faecalis observed in the presence of both GT and vancomycin (4 µg/ml). As well as zinc chelation, DTG also appears to chelate Cu2+, but not Mn2+ using a 4-(2-pyridylazo)resorcinol assay system and Zn2+ as a positive control. DTG also specifically reacts in Fe3+-containing Siderotec™ assays, most likely by Fe3+ chelation from test reagents. GSH or DTT show no activity in these assays. Confirmatory high resolution mass spectrometry, in negative ion mode, confirmed, for the first time, the presence of both Cu[DTG] and Fe[DTG]2 chelates. Label free quantitative proteomic analysis further revealed major intracellular proteomic remodelling within E. faecalis in response to GT exposure for 30–180 min. Globally, 4.2–7.2% of detectable proteins exhibited evidence of either unique presence/increased abundance or unique absence/decreased abundance (n= 994–1160 total proteins detected), which is the first demonstration that GT affects the bacterial proteome in general, and E. faecalis, specifically. Unique detection of components of the AdcABC and AdcA-II zinc uptake systems was observed, along with apparent ribosomal reprofiling to zinc-free paralogs in the presence of GT. Overall, we hypothesis that GT-mediated bacterial growth inhibition appears to involve intracellular zinc depletion or reduced bioavailability, and based on in vitro chelate formation, may also involve dysregulation of Cu2+ homeostasis.

Item Type:	Article
Keywords:	Antibacterial activity; Antimicrobial resistance; Bacteria; Bioavailability; Chelates; Chelation; Copper;
Academic Unit:	Faculty of Science and Engineering > Biology Faculty of Science and Engineering > Research Institutes > Human Health Institute
Item ID:	18973
Identification Number:	https://doi.org/10.1038/s41598-023-43300-w
Depositing User:	David Fitzpatrick
Date Deposited:	03 Oct 2024 12:43
Journal or Publication Title:	Scientific Reports
Publisher:	Nature Publishing Group
Refereed:	Yes
URI:	Publisher
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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