Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion depletion

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 hypothesise 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.

www.nature.com/scientificreports/which comprises an essential intramolecular disulphide bridge, which can be converted to the dithiol form and exhibit potent Zn 2+ chelation properties 11,12 .In addition to showing potent cytotoxicity against eukaryotic cells, GT has been shown to display broad spectrum antibiotic properties 13,14 .The accepted hypothesis has been that the oxidation and reduction of the disulphide bridge, which are key to ETP activity, produces cytotoxic reactive oxygen species 15 .
The growth inhibitory effects and mechanism of action of GT have mainly been studied in animal cells and in fungi [16][17][18][19][20][21] .It is now clear that upon entry into animal cells GT is reduced to dithiol gliotoxin (DTG) via cellular GSH, this results in ROS generation, depletion of intracellular GSH and GT efflux from cells 13 .Cell death ultimately follows.No information is available on the specific and direct impact of DTG in animal cells, although it has been demonstrated to directly inhibit the activity of LTH 4   12   .In fungi which produce GT (e.g., A. fumigatus) and related ETPs, a self-protection system involving DTG oxidation to GT via oxidoreductase GliT and efflux via transporter GliA, is present [22][23][24][25] .If levels of GT/DTG increase beyond the capacity of the oxido-efflux system, then DTG is bis-thiomethylated to an inactive form by bis-thiomethyltransferase GtmA 26,27 .Additionally, many fungi lacking ETP biosynthetic capacity possess a gtmA ortholog, possibly to dissipate environmentally acquired GT or other ETPs 18,26 .DTG chelates Zn 2+ and in A. fumigatus, it is now clear the GT is produced in low Zn 2+ environments 11,28,29 .Although the precise relationship between GT/DTG and Zn 2+ in fungi remains to be elucidated, elevated DTG levels can deplete intracellular Zn 2+ , possibly inhibit intracellular metalloenzymes and inhibit growth 11,30 .There is some evidence that fungal Zn 2+ acquisition systems may be activated under these conditions 11,31 .Reduced holomycin, a functional dithiolopyrrolone (DTP) analogue of DTG, has been shown to inhibit bacterial metallo-β-lactamases (MBL) and proposed to alter bacterial metal homeostasis 32 .
Although there is a plethora of publications describing the inhibitory activity of GT and other ETPs against bacteria 33,34 , to the best of our knowledge, there is limited data on the GT mechanism of inhibitory action against bacterial species.Indeed, apart from a publication inferring interference with GSH levels 35 , no mechanistic details have been forthcoming about how GT inhibits bacterial growth.Interestingly, it has been demonstrated 14 that while GT inhibited growth of a range of bacterial species and interfered with biofilm formation, culture supernatant from A. fumigatus ΔgliG, deficient in GT biosynthesis 36 , exhibited no antimicrobial activity.It has also been revealed that in addition to inhibiting growth of Staphylococcus aureus, GT augments the activity of vancomycin, thereby revealing synergistic activity, of unknown origin, between both antimicrobial agents 37 .Thus, although GT and other ETPs have been shown to inhibit bacterial growth, often at low µg/ml concentrations, there is a real dearth of information on how bacterial growth is inhibited.
It is not unreasonable to speculate that upon entry into bacterial cells that GT is reduced by unknown cellular reductants and that it is the reduced (DTG) form of GT which effects growth inhibition 38 .Given that Zn 2+ chelation by DTG has been observed 11,12 , we speculate that this could also occur in bacteria, along with possible Zn 2+ ejection from bacterial enzymes.We further hypothesise that depletion of intracellular Zn 2+ levels would be predicted to activate uptake systems and metalloenzyme activity/stability, or abundance, could be adversely affected by GT/DTG-mediated Zn 2+ -ejection, in part as previously speculated 39 .This manuscript describes work to explore the effect of the non-ribosomal peptide GT on the Gram-positive bacterial species E. faecalis.Specifically, this work aims to decipher the mechanism of action behind the inhibitory activity of GT against bacteria.It is framed in the context of the reduced form of GT, dithiol gliotoxin (DTG), and its demonstrated ability to chelate zinc (Zn [DTG]).

GT inhibits Gram positive and negative bacterial growth, which is relieved by zinc addition
Both Gram positive species were susceptible to growth inhibition in the presence of GT.Specifically, E. faecalis showed significant and dose-dependent growth inhibition when grown in the presence of gliotoxin (0.9375-60 µM; p < 0.05-0.0001).Addition of Zn (10, 100, or 200 µM) relieved most of this inhibition at 10 µM and above Zn supplementation (p < 0.0001) (Fig. 1A).Cu supplementation (10, 100, or 200 µM) relieved some of this inhibition at as little as 10 µM Cu and total relief was seen at 100 and 200 µM Cu supplementation (p < 0.0001) (Fig. 1B).Addition of Mn (10, 100, or 200 µM) did not relieve growth inhibition (Fig. 1C).E. faecium showed significant and dose dependent growth inhibition when grown in the presence of gliotoxin (0.9375-60 µM) (optimally p < 0.0001).Zn (10, 100, or 200 µM) addition showed some relief of inhibition from 10 µM Zn and total relief was seen at 100 and 200 µM Zn supplementation (p < 0.0001) (Fig. 1D).Cu (10, 100, or 200 µM) also relieved growth inhibition at as little as 10 µM Cu.Total relief was seen at 100 (p < 0.005) and 200 µM Cu (p < 0.0001) supplementation (Fig. 1E), however, Mn addition (10, 100, or 200 µM) did not relieve GT-mediated growth inhibition (Fig. 1F).In E. faecalis, we interpret relief of GT-mediated inhibition requiring higher Cu concentration as indicative of differential biosystem dependency on this metal ion.E. faecalis V4932 has previously been shown to be sensitive to A. fumigatus GT-containing culture supernatant 33 , in accordance with our observations.As far as we can ascertain, there is only a single report of GT-mediated growth inhibition of E. faecium, or associated metal relief of same, in the literature 40 .Future work will be required to elucidate which Gram positive bacterial systems are subject to metallo-mediated GT inhibition.
Both Gram negative species were also susceptible to GT-mediated growth inhibition.K. pneumoniae showed significant and dose-dependent growth inhibition at GT concentrations ≥ 30 µM (p < 0.0001).Addition of Zn (10, 100, or 200 µM) relieved most inhibition at as little as 10 µM Zn, with near total relief evident at 100 and 200 µM Zn supplementation (p < 0.05-0.0001)(Fig. 2A).Cu (10, 100, or 200 µM) relieved almost all this inhibition (p < 0.05-0.0005)(Fig. 2B), yet addition of Mn (10, 100, or 200 µM) showed no relief of inhibition (Fig. 2C).Of all bacterial species tested, growth of A. baumannii was most significantly inhibited in a dose-dependent manner in the presence of GT (≥ 30 µM) (p < 0.0001).Addition of Zn at 10 µM relieved almost all this inhibition in the presence of 30 µM GT (p < 0.0001) but provided no relief at 60 µM GT.Near total relief was seen at 100 and 200 µM Zn supplementation (p < 0.0001) (Fig. 2D).Cu at as low as 10 µM also relieved almost all growth inhibition (p < 0.0001), at 30 µM GT, but provided no relief at 60 µM GT.Near total relief was seen at 100 and 200 µM Cu supplementation (p < 0.0001)(Fig.2E).As with all other species, Mn (10, 100, or 200 µM) did not relieve GTmediated growth inhibition in A. baumannii (Fig. 2F).Experimentation showed no significant (p < 0.05) relief of GT-induced inhibition with supplemental Fe addition at any concentration to E. faecalis (Supplementary Fig. 1).This leads to the conclusion that either there is sufficient Fe present in the TSB media (5.37 µM) 41 to overcome GT inhibition or that intracellular DTG preferentially chelates Zn or Cu.This preferential chelation may be due to the need for two DTG molecules per Fe ion to form the Fe[DTG] 2 complex as opposed to one for Zn and Cu.ICP-MS or similar technology could be used to gain insight into the intracellular concentrations of Fe ions when the cells are challenged with GT/DTG or protected from challenge with supplementary Zn or Cu, and this will form the basis of future detailed work.The OD600 values equate to the following CFU/ml for E. faecalis: 0.2 OD600 = 1.2 × 10 8 CFU/ml and 0.1 OD600 = 7 × 10 7 CFU/ml.Significant inhibition due to GT exposure is indicated with black asterisks (* = P < 0.05, ** = P < 0.01, *** = P < 0.0001).Significant relief of inhibition at any metal concentration is shown with red asterisks (* = P < 0.05).
E. faecalis cultures grown in the presence of GT (5 µg/ml) for 1 h contained 12.6% less Zn than MeOH treated counterparts, as measured by zinquin fluorescence.To our knowledge this is the first demonstration that GT/ DTG alters the internal metal abundance in bacteria, which suggests that the metal-chelating action of DTG has a negative effect on cellular growth and that GT may not be the actual active agent.This apparent Zn depletion likely plays a significant role in the inhibitory effects of GT/DTG and explains why Zn supplementation ameliorates the effects of GT.However, we observed no significant difference between intracellular Zn levels in the presence or absence of GT at 5, 15 and 30 min GT exposure which suggests that it is the bioavailability of Zn, as opposed to total intracellular Zn, which may mediate the bacterial response to GT (Supplementary Fig. 2).Importantly, the 30 min time-point overlaps with the quantitative proteomic experimentation where increased abundance of the Zn uptake receptor system is observed.This leads us to hypothesise the GT is reduced to DTG upon cellular uptake, which in turn chelates intracellular Zn thereby generating a Zn-limiting environment  www.nature.com/scientificreports/which activates the Zn uptake system.Future work will be focused on identifying intracellular Zn-DTG chelate and using ICP-MS to absolutely quantify intracellular Zn levels, following chelate formation and purification.Relevantly, a range of zinc chelators, including TPEN, were evaluated for their ability to resensitize metalloβ-lactamase (MBL)-producing bacteria to antibiotics 42 .Synergistic activity between TPEN and meropenem was observed against K. pneumoniae, Chryseobacterium indologenes, Elizabethkingia meningoseptica and Stenotrophomonas maltophilia in in vitro experiments.Moreover, using the Galleria mellonella in vivo infection model, chelators TPEN or nitroxoline in combination with meropenem resulted in increased larval survival following infection with either K. pneumoniae, E. meningoseptica or S. maltophilia 42 .Thus, it is clear that artificial and naturally-occurring zinc chelators have potential to either prevent microbial growth or inactivate MBLs to overcome AMR.The demonstration of GSH-mediated activation of holomycin to reduced holomycin, with zinc-chelating ability, further underscores this biomedical application 43 .

GT is bactericidal at high concentrations
An antibacterial agent is defined as bactericidal if it reduces the CFU/ml of a culture by more than a 3 log 10 -fold decrease.At 8 h, all GT concentrations show bactericidal activity against E. faecalis with a 3 log 10 -fold decrease (Supplementary Fig. 3).At 24 h, a dose-dependent recovery of the bacterial cells was observed where GT (1.875 µM) was no longer sufficient to inhibit growth.Both GT (3.75 and 7.5 µM) maintained their efficacy.GT (3.75 µM) was still effective though it fell slightly below the 3 log 10 -fold decreased cut off with 6.4 × 10 7 CFU/ml compared to the control cultures 1.05 × 10 10 CFU/ml.However, the highest GT concentration (7.5 µM) maintained bactericidal activity with 2.12 × 10 5 CFU/ml.

Zinc relieves chelator-mediated (GT and TPEN) growth inhibition of E. faecalis in a dose-dependent manner
To show that metal chelation is a viable mechanism of bacterial growth inhibition, and that Zn supplementation relieves growth inhibition, the known Zn chelator TPEN was used as GT comparator.Indeed, our data shows that both molecular species chelate Zn whereby we determined the GT Zn pKd = 8.72 and TPEN Zn pKd = 9.69, respectively (Supplementary data).GT and TPEN (0-60 µM) were used to treat cultures of E. faecalis with and without Zn supplementation.When E. faecalis was grown in the presence of GT there was insignificant inhibition of growth (< 5%) at low concentrations (< 3.75 µM).The effects of GT increased with the dosage showing minor (6.1%) but significant (p = 0.0001) growth inhibition at 7.5 µM GT.The most significant (p < 0.0001) inhibition was seen at 15-60 µM GT.Inhibition rapidly increased at these concentrations 15 µM (8.0%), 30 µM (10.0%), and 60 µM (65.9%).Supplementation of 60 µM GT treated samples with all Zn concentrations showed significant (p < 0.0001) relief of inhibition with the percentage growth relative to the 0 µM Zn treated samples increasing by 53.3-55.8%(Supplementary Fig. 4).When E. faecalis was grown in the presence of TPEN significant (p = 0.0167) growth inhibition (5.1%) was seen at 7.5 µM TPEN, which became more significant (p = 0.0005) at 15 µM TPEN with slightly greater growth inhibition (6.9%).The most significant (p < 0.0001) growth inhibition was seen at 30 µM (80.1%) and 60 µM (91.5%)TPEN.Zn supplementation showed significant (p < 0.0001) relief of growth inhibition (70.0-71.0%).At 60 µM TPEN, 10 µM Zn was no longer able to provide significant (p > 0.05) relief of growth inhibition.However, 100 and 200 µM Zn supplementation provided significant (p < 0.0001) relief of inhibition, reducing growth inhibition by 83% (Fig. 3A).These data provide strong evidence that GT inhibits growth through intracellular Zn limitation similar to TPEN.Moreover, it is in accordance with previous work that GT/DTG, acts as a zinc chelator and either binds free zinc, or ejects it from metalloenzymes in fungi 11,29 .TPEN is a well-characterised zinc chelator, and it too appears to inhibit E. faecalis growth in a dose-dependent manner, which is also completely reversible by zinc addition (Fig. 3A).Combined, these data demonstrate, for the first time, a potential mechanism(s) of action, involving disruption of intracellular zinc homeostasis, for GT/ DTG-mediated bacterial growth inhibition.Moreover, it complements the work of others who have deployed a range of zinc chelators (e.g., TPEN, reduced holomycin, D-alanyl-D-alanyl-D-alanine methyl ester functionalized N,N,N'-tris(2-pyridylmethyl)-ethylenediamine and tris-picolylamine (TPA) to overcome MBL degradation of target antibiotics 32,42,44,45 .Conversely, in pioneering work, microarray analysis of E. faecalis exposure to Zn and Cu revealed that two modules were implicated in the microbial response to metal exposure 46 .So-called module I and II, comprised genes implicated in Zn homeostasis (including the Zur transcription factor) and those involved in stress responses/basal metabolism, respectively.Relevantly, exposure to Zn resulted in downregulation of module I-encoded genes (adcABC/adcA-II) responsible for the Zn uptake system in E. faecalis 46 .

GT augments vancomycin-mediated growth inhibition of E. faecalis-which is relieved by Zn 2+
Vancomycin has been shown to transiently increase expression of selected Zur regulon genes in Streptomyces coelicolor and also to bind to zinc in vitro 47 .Moreover, zinc has been demonstrated to induce vancomycin polymerisation thereby leading to enhanced antibiotic activity against E. faecalis 48 .Of further relevance is that it has been independently shown that GT acts synergistically with vancomycin, fusidic acid and linezolid, to inhibit Staphylococcus aureus growth 37 , an observation which suggests differential mechanistic actions and strongly underpins the rationale for further investigating that of GT/DTG.Interestingly, this previous work did not suggest a metal-dependent basis of GT-mediated inhibition 37 .However, herein we have observed that GT/DTG augments the vancomycin-mediated inhibition of E. faecalis growth, and that zinc addition (200 µM) negates the observed combinatorial inhibition (Fig. 3B, C).We show that GT inhibits the growth of E. faecalis in a dose-dependent manner, and that this inhibition is readily relieved by the addition of zinc.This implicates the disruption of microbial zinc homeostasis as a mechanism of GT antimicrobial action and provides an insight into microbial systems targeted by GT.This inhibitory effect was further explored in combination with vancomycin, the antibiotic of last resort.Checkerboard assays using GT and vancomycin showed that the presence of sub-inhibitory concentrations of GT (5 and 10 µg/ml) were sufficient to significantly (p < 0.03) lower the MIC of vancomycin against E. faecalis.This combinatorial work has also shown that GT may inhibit biofilm formation as there were apparent differences in biofilm appearance between shaking and static incubation.A unique speckled phenotype was seen at vancomycin (4 µg/ml) which was diminished with increasing GT.The presence of Zn (200 µM) attenuated the effects of GT on this phenotype (Fig. 3D).Interestingly, although GT (0.15-10 µg/ml) impeded biofilm formation in the presence of vancomycin (4 µg/ml), this inhibition did not reach statistical significance (Supplementary Fig. 5).Thus, alternative approaches will be required to further dissect any combinatorial or synergistic activity of both antimicrobials and the role of zinc in inducing vancomycin polymerisation to augment its antimicrobial activity 48 .

DTG chelates zinc, copper and iron
Using the colorimetric metal chelator 4-(2-pyridylazo)resorcinol (PAR), it was shown that DTG exhibits copper chelation properties (Fig. 4A-D).Specifically, DTG-mediated liberation of Zn (positive control; 11 ) and Cu from respective PAR complexes was determined using a decrease in absorbance at 495 nm and 513 nm, respectively (Fig. 4A,C).A dose-dependent decrease in Zn(PAR) 2 and Cu(PAR) 2 complexes corresponding to an increase in DTG concentration was observed, indicating that DTG competitively liberates both Zn and Cu from PAR (Fig. 4A,C).Under test conditions, DTG (120 µM) dissociated most, but not all, Zn from Zn(PAR) 2 (Fig. 4A).In contrast, TPEN removed all Zn from Zn(PAR) 2 at concentrations ≥ 80 µM (Fig. 4B).Interestingly, DTG was more effective at dissociating Cu from Cu(PAR) 2 with the vast majority dissociated at 80 µM and complete dissociation observed at ≥ 100 µM (Fig. 4C).DTG was only slightly less effective than TPEN at dissociating Cu from PAR, with ≥ 80 µM TPEN resulting in dissociation of all Cu from Cu(PAR) 2 (Fig. 4D).DTG does not remove Mn from PAR chelates or Mn does not form chelates with PAR (data not shown), in accordance with previous observations regarding absence of Sporidesmin A:Mn chelates 49 .It is important to note that the formation of DTG:Zn and DTG:Cu chelates is in accordance with the observation that GT-mediated bacterial growth inhibition is reversed by these metals and not Mn, which does not appear to form a metal chelate with DTG.
The colorimetric Siderotec Total assay contains a Fe 3+ chromophore.DTG (4.7-304 µM) appeared to dissociate Fe from the chromophore in a dose-dependent manner culminating in 76% Fe removal at DTG (304 µM) after 10 min (Fig. 5A).Relevantly, neither GT or TCEP revealed any ability to dissociate Fe from the chromophore in the Siderotec Total™ assay (Fig. 5A).Furthermore, the ability of DTG to interact with Fe was also assessed using a fluorescence-based Siderotec HiSens™ assay, which confirmed a dose-dependent removal of Fe from the constituent fluorophore with increasing DTG (4.7-304 µM) (Fig. 5B).In this assay only 25% Fe was removed at 304 A.  µM DTG, however, a temporal effect was apparent with DTG-mediated dissociation of increased amounts of Fe over time (41% Fe dissociation after 60 min, decreasing to 37.6% after 120 min at 300 µM DTG) (Fig. 5C).Again, neither GT or TCEP (4.7-300 µM) showed any notable impact on the assay at any time point, though TCEP did show slightly higher fluorescence signal than GT (Fig. 5D,E).Moreover, compared to DTG, neither GSH or DTT showed any impact on fluorescent chelate-Fe 3+ stability in the Siderotec-HiSens™ assay (Supplementary Fig. 8), indicating the specificity of DTG for Fe 3+ displacement from fluorescent chelates.High resolution mass spectrometry in negative mode confirmed the presence of DTG:Zn (positive control), DTG:Cu, and (DTG) 2 :Fe chelates, and the resultant spectra compared to those from theoretical chelate structures (Fig. 6).The DTG:Zn chelate stoichiometry was known from previous work 11 who found the metal adduct had an m/z = 424.93851,which corresponded to a 1:1 complex of DTG:Zn with a Cl − ion.This structure was herein identified to exhibit m/z = 424.9393([DTG(Zn)-2H + Cl] − )(Fig.6A), in accordance with previous data.The DTG:Cu chelate revealed m/z = 423.9397,corresponding to a 1:1 DTG:Cu chelate with Cl -present ([DTG(Cu)-2H + Cl] − )(Fig.6B) and the (DTG) 2 :Fe chelate showed an m/z = 708.0164which corresponds to a 2:1 complex of DTG:Fe with no Cl -adduct present (Fig. 6C), but with an overall single negative charge suggesting Fe 3+ as the iron cation ([DTG 2 (Fe)-2H] − ).
Overall, the revelation that DTG can chelate Cu and Fe is of significant biological interest, and to our knowledge has not previously been observed.It is notable that DTG:Mn chelates were not observed and suggests an element of specificity regarding the formation of DTG:Zn, DTG:Cu and (DTG) 2 :Fe chelates.Although outside the scope of the present work, the potential interaction between DTG and Fe may be of particular relevance in A. fumigatus and other fungi which biosynthesise ETP-type biomolecules as it suggests an additional rationale for controlling intracellular levels of DTG to avoid Fe 3+ reduction and disrupted redox homeostasis.In E. faecalis, GT exposure at T = 60 min resulted in the loss of detection of ferrous transport protein A which indicates metal ion remodeling may occur due to hitherto unidentified effects of GT/DTG in bacteria.It is notable that gliT expression in A. fumigatus was reduced upon deletion of SreA, a transcriptional repressor of siderophore biosynthetic enzymes transporters to avoid excess iron uptake and resultant toxicity under iron-replete conditions 50 .This observation, in combination with our demonstration of (DTG) 2 :Fe chelate formation, suggests that GliT may potentially contribute to Fe 2+ homeostasis in A. fumigatus wild-type.However, further work is undoubtedly required to fully dissect the biological significance of these observations.When a nucleophile donates lone-pair (n) electron density into the empty π* orbital of a nearby carbonyl group, it is referred to as an n → π* interaction 51 .In ETPs, the n → π* interactions can decrease the disulphide reduction potential, which may confer biologically-relevant stability on the disulphide bond in physiological environments.Indeed, it has been proposed that the two strong n → π* interactions in an ETP, like GT, can nearly completely compensate for the molecular instability caused by the strained conformation of the disulphide bond 52 .Moreover, intramolecular stability may also be affected by the hydrophobicity of the environment in which the molecule is located.Thus, we speculate that the intracellular bacterial environment is compatible with GT reduction and consequent metal ion chelation.

GT causes significant remodelling of the E. faecalis proteome
Extensive alterations, including protein unique presence and increased abundance as well as unique absence and decreased abundance were observed in the E. faecalis proteome following GT addition (5 µg/ml) for 30, 60 and 180 min respectively (Tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).Overall, between T = 30-180 min GT exposure, 4.2-7.2% of detectable proteins exhibited evidence of either unique presence/increased abundance or unique absence/decreased abundance, where total proteins detected ranged from 994 to 1160 across all analyses.To our knowledge, this is the first demonstration that GT affects the bacterial proteome in general, and E. faecalis, specifically.
Three ribosomal proteins are either uniquely present or increased in abundance following GT addition, and two Zn 2+ uptake membrane proteins (AdcA and AdcC of the Gram positive AdcABC system), out of 32  www.nature.com/scientificreports/proteins in this dataset, were significantly increased in abundance (log 2 -fold 2.4-7.28) at 30 min (Tables 1, 2; Supplementary Fig. 9).This is the first demonstration that GT affects bacterial ribosomal protein presence, as has been predicted 39 .Increased Zn 2+ uptake transporter abundance clearly suggests that intracellular Zn 2+ may be complexed by GT/DTG resulting in a limiting intracellular environment.Our observations complement previous work which revealed significantly increased adcABC and adcA-II gene expression following exposure to 5 µM TPEN 46 .Moreover, it is in complete accordance with our data which shows that Zn 2+ relieves GT-mediated growth inhibition and that GT/DTG is a Zn 2+ chelator.It is further conceivable that DTG may eject Zn 2+ from intracellular proteins resulting in inactivity and possibly denaturation.The increased abundance of the Zn 2+ uptake membrane proteins was also evident at T = 60 and they were uniquely detected at T = 180 min post-GT addition (Tables 5, 6, 9, 10).Our hypothesis that GT-mediated zinc depletion (reduced bioavailability) was primarily responsible for quantitative proteomic alterations, was investigated by assessment if Zn co-addition (200 µM) for 30 min with GT Table 1.Proteins which are uniquely present in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 30 min in the log phase (0.3-0.4 OD600).*This protein exhibited overall increased abundance > 1.5-fold and was only detectable in 1 of 3 untreated samples.www.nature.com/scientificreports/would reverse the observed GT-induced proteomic alterations.This chemo-complementation approach revealed that 1 of 16 proteins uniquely detected by GT addition only exhibited reversed abundance in the presence of GT and Zn, of the 12 proteins uniquely absent with GT, 3 were decreased in abundance in the presence of Zn while 1 (Q834B2) was uniquely detected in the Zn supplemented samples (Supplementary Tables 4 and 5).After 30 min exposure to GT (5 µg/ml) and Zn (200 µM), there was a minor and insignificant increase in AdcA, AdcC, and AdcA-II when compared to GT (5 µg/ml) only.This data indicates that 30 min may be too short a time frame to see a complete disappearance of the AdcABC system proteins.After 60 min, AdcA was decreased in abundance (− 1.5 log2 fold change), AdcC also exhibited decreased abundance (− 1.3 log2 fold change) (although not statistically significant (p < 0.5)) and AdcA-II was also insignificantly decreased in abundance (p < 0.2; − 1.3 log2 fold change) in the presence of GT (5 µg/ml) and Zn (400 µM), when compared to GT (5 µg/ml) only (Supplementary Table 6).In combination, these data indicate that addition of Zn (400 µM) relieves some of the effects of GT (5 µg/ml), but was either insufficient to result in a rapid decrease in the requirement for the Zur-regulated zinc uptake system or 60 min incubation was sub-optimal for this comparative study.Hence, future work will focus on evaluating and defining the optimal conditions of zinc reversal of significantly elevated AdcABC and Adc-II uptake systems in E. faecalis.However, the increased abundance or unique presence of E. faecalis zinc import proteins (AdcA and C) in the presence of GT at all timepoints is in complete accordance with the independent demonstration that disruption of orthologous genes encoding AdcA or AdcB and AdcC, components of the zinc uptake system in Streptococcus mutans, caused severe growth inability under zinc-deplete conditions 53 .Furthermore, it was demonstrated that the S. mutans ΔadcBC mutant exhibited a severe colonisation defect in a rat infection model system 53 .In a further study in E. faecalis, it has been elegantly shown that AdcABC and adhesion lipoprotein AdcA-II function in a cooperative manner to ensure Zn homeostasis.Deletion of system components resulted in zinc-associated growth defects and increased sensitivity to antibiotics which target the bacterial cell wall 54 .These authors also showed that bacterial virulence was attenuated in zinc transporter deletion mutants and propose interference with high affinity zinc importers as a potential therapeutic strategy to combat E. faecalis infection 54 .In combination, these deletion studies and associated identification of attenuated virulence in the absence of zinc uptake systems underpin the strategy and utility of using GT/DTG couple as a means of identifying new antibacterial drug targets 38 .Interestingly, an E. faecalis pyridine nucleotide-disulphide oxidoreductase (PNDO)(H7C719) was uniquely detected in the presence of GT (Table 1 and 5) at T = 30 and 60 min exposure.To our knowledge, this is a protein of unknown function in E. faecalis and its presence is suggestive of altered redox homeostasis upon Table 3.Proteins which are uniquely absent in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 30 min in the log phase (0.3-0.4 OD600).*This protein exhibited overall decreased abundance > 1.5-fold and was only detectable in 1 of 3 treated samples.GT exposure.It is also relevant that the self-protection enzyme gliotoxin oxidoreductase GliT in A. fumigatus converts DTG to GT 22,23 and is also classified as a PNDO-type enzyme.Future work will involve targeting this bacterial PNDO for deletion and mutant characterisation.The unique or elevated abundance of specific ribosomal proteins ((T30 = 50S ribosomal protein L33 4 (P59629), 30S ribosomal protein S14 1 (Q8KU58), and 30S ribosomal protein S15 (Q82ZJ1); T60 = 50S ribosomal protein L33 4 (P59629), 50S ribosomal protein L28 (Q82ZE4), 30S ribosomal protein S14 1 (Q8KU58); T180 = 50S ribosomal protein L33 4 (P59629), 30S ribosomal protein S14 2 (Q82Z70); (plus uniquely absent 50S ribosomal protein L33 3 (P59628)) was also observed (Supplementary Fig. 10 and Supplemental Table 1).These changes in ribosomal protein abundance were indicative of a shift towards non-zinc binding paralogs upon GT addition, which is in accordance with the proposal of Danchin that the prokaryotic ribosome may act as a zinc store 39 .Thus, when GT is added to E. faecalis, we hypothesise that resultant zinc depletion would lead to zinc release from ribosomes and consequent increased presence of selected zinc-free ribosomal protein paralogs.Table 5.Proteins which are uniquely present in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 60 min in the log phase (0.3-0.4 OD600)..In the work of Dow et al., non-zinc-binding paralogs were identified as functional replacements for Zn 2+ -dependent paralogs and were involved in the transcriptomic response to Zn 2+ -limitation.
GT uptake and predicted conversion to DTG by intracellular reduction by GSH, L-Cys or other reductants would likely lead to dissipation of these metabolites and increased biosynthesis of same.In accordance with this prediction, a cysteine synthase (Q834Q6) was detected as significantly increased in abundance (p < 0.025) at T = 180 min, with high confidence given that 77% sequence coverage was evident (Tables 9,10).This enzyme may result in either L-Cys or ultimately GSH formation in E. faecalis.Importantly, this proteomic observation aligns with the detection of higher (+ 38%) total intracellular thiol concentration (0.156 nmol per mg cells) following GT addition to E. faecalis.Detection of a putative cysteine synthase B (Q838Z3) as significantly decreased in abundance (p < 0.02) at T = 30 min requires future analysis (Tables 3, 4).Table 6.Proteins which are increased in abundance in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 60 min in the log phase (0.3-0.4 OD600).*This protein was found in 2/3 GT treated samples and 1/3 untreated controls which prevented calculation of a P-value.Table 7. Proteins which are uniquely absent in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 60 min in the log phase (0.3-0.4 OD600).Table 8.Proteins which are decreased in abundance in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 60 min in the log phase (0.3-0.4 OD600).www.nature.com/scientificreports/Detection of a cobalamin synthesis protein (Q82Z69) at T = 30 and 60 only post-GT addition (Tables 1, 2, 5, 6) suggests that vitamin B 12 biosynthesis is increased in E. faecalis due to GT presence.Allied to this is the increased abundance of Met sulphoxide reductase MsrB (P0DM32) which was identified in 2/3 GT treated samples versus 1/3 controls therefore precluding the calculation of a p-value (Tables 5, 6).This enzyme has been shown to contribute to E. faecalis response to oxidative stress, by L-Met regeneration, and virulence 56 .The detection of both these enzymes, along with significantly increased abundance (p < 0.03) of thioredoxin (Table 1, 2) suggests that the GT effect on E. faecalis also involves disrupted L-Met oxidation, redox homeostasis and possibly Methyl/ Met cycle due to SAM requirements.Future work will investigate the altered abundance of enzymes involved in L-Met metabolism by targeted deletion studies, since few data is currently available on these phenotypes.
Our quantitative proteomic analyses also revealed absence or significantly reduced abundance of components of the E. faecalis PTS (phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system), mainly PTS system: IIA component, mannose-specific EIIAB and IIC components between 30 and 180 min post-GT addition, though one protein in this system, PTS System, beta-glycoside-specific IIABC component was uniquely present with GT (Q831B4) (Tables 1, 3, 4, 7, 8, 11, 12).This system has recently been shown to repress ribosomal biosynthesis and to increase the sensitivity of E. faecalis to gentamycin.Overexpression of PTS also increased the sensitivity of E. faecalis to the antibiotic, daptomycin 57 .Interestingly, alkaline stress caused reduced abundance of PTS system components in E. faecalis V583 as determined by proteomic analysis 58 .Our observation of decreased abundance of PTS system components, in response to GT presence is in accordance with these studies which suggests that deactivation of the PTS system reduces the impact of antibacterial compounds (e.g., daptomycin, GT or gentamycin) or conditions (alkaline stress).Table 9.Proteins which are uniquely present in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 180 min in the log phase (0.3-0.4 OD600).*This protein exhibited overall increased abundance > 1.5-fold and was only detectable in 1 of 3 untreated samples.Table 10.Proteins which are increased in abundance in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 180 min in the log phase (0.3-0.4 OD600).Through proteomic analysis we show that GT may affect intracellular metal ion homeostasis, possibly by interference with intracellular zinc pools in E. faecalis.Proteins associated with key pathways, several of which are controlled by the zinc-dependent Zur transcription factor 46 , were altered in abundance when E. faecalis was exposed to GT.This metallo-centric model implicates DTG as the active form of GT and supplants the conventional perspective of altered redox homeostasis due to GT presence as the sole factor responsible for the antimicrobial activity of GT/DTG.In conclusion, this work illustrates the GT/DTG couple potential for providing new ways to combat the growing antibiotic resistance threat as well as yielding new mechanistic approaches for treating antibiotic resistant infections 38 .
In summary, DTG causes Zn 2+ and Cu 2+ to specifically dissociate from PAR in a concentration-dependent manner.DTG can specifically strip or remove Fe 3+ from low and high sensitivity Fe 3+ chelates in a concentrationdependent manner.Notably, this interaction is also time-dependent.The reducing agent DTT and antioxidant GSH had no effect on fluorescent chelate:Fe 3+ stability, implicating chelation as the mechanism of Fe 3+ removal from this chelate by DTG.When exposed to GT (5 µg/ml) E. faecalis showed significant and specific proteome alterations, many of which are linked to the occurrence Zn 2+ -limiting or bioavailability conditions; and for the first time imply that GT/DTG induce zinc starvation within E. faecalis.DTG may chelate free Zn 2+ and eject it from intracellular metalloproteins, thereby causing growth inhibition.During manuscript revision a report emerged which also addressed the role of gliotoxin as an anti-bacterial agent 59 .Table 11.Proteins which are uniquely absent in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 180 min in the log phase (0.3-0.4 OD600).*This protein exhibited overall decreased abundance > 1.5-fold and was only detectable in 1 of 3 treated samples.These cultures were incubated overnight (37°C; 170 rpm).The OD600 nm of the cultures were determined and aliquots of each culture were diluted to OD600 nm = 0.2 using TSB.Cultures were added to wells (100 µl/well) of 96-well plates, in triplicate, containing 100 µl TSB supplemented with GT (0-120 µM) and Zn, Cu, or Mn (0, 20, 200, or 400 µM), incubated (37°C; Static; 18 h) and OD600 nm determined using a BioTek Synergy HT 96-well plate reader.Following GT exposure (GT: 0-60 µM), iron supplementation (0, 10, 100 and 200 µM) was carried out for E. faecalis only.TSB has been shown via ICP-AES 41 to basally contain: Zn: 0.6 ppm = 9.177 µM; Cu: < 0.1 ppm = < 1.5737 µM and Fe: 0.3 ppm = 5.372 µM.Data analysis was performed using GraphPad Prism 9.5.0 via two-way ANOVA.

Zn determination by zinquin method
E. faecalis (ATCC 19433) was grown overnight in TSB (37 °C 170 rpm).These cultures were used to inoculate fresh TSB to an OD600 of 0.05 until reaching an OD600 of 0.3-0.4.The cultures were divided into even aliquots and spiked with GT (5 µg/ml final) or MeOH.The cultures were returned to the incubator (37 °C 170 rpm) and 15 ml aliquots were taken after 5, 15, and 30 min.The assay was also performed for 60 min.Aliquots were centrifuged 4500 g at 4 °C for 10 min and the supernatants discarded.Pellets were washed extensively in PBS and transferred to 1.5 ml tubes.Samples were centrifuged 15,000 g for 10 min at 4 °C.After weighing pellets, 400 µl PBS (0.03% (w/v) albumin from chicken egg white) was added to each sample and vortexed into solution followed by sonication on ice (6 times MS72 probe, power ≤ 20%, cycle 6) and transfer to black 96 well plates (200 µl per well) for addition of 3.5 µl Zinquin in DMSO (12 mM stock).After pipette-mixing and incubation (37 °C in the dark for 40 min.),Zn levels were calculated by comparison to a Zn standard curve.All dilutions were done in PBS (0.03% albumin from chicken egg white).Fluorescence detection: excitation: 360/40 nm, emission: 460/40 nm, Gain: 65.

Gliotoxin x vancomycin biofilm evaluation by crystal violet assay
E. faecalis cultures (0.2 OD600) were added to 96-well plates, 100 µl/well, containing 100 µl TSB supplemented with GT (0-10 µg/ml) and vancomycin (4 µg/ml) followed by incubation (37 °C; Static), 18 h and OD600 determined using a BioTek Synergy HT 96-well plate reader.The media was removed from the wells, washed with deionised H 2 O then dried.Crystal violet (0.1% (w/v)) was added to each well, incubated for 10 min at room temperature, discarded, the wells washed with deionised H 2 O and then dried.Acetic acid (30% (v/v)) was added to each well and allowed to incubate for 10 min at room temperature.The wells were pipette-mixed then read at 595 nm using a BioTek Synergy HT 96-well plate reader.

GT time-kill assay
Three colonies of E. faecalis were used to inoculate TSB followed by incubation overnight (37 °C; 170 rpm).The OD600 of the cultures were measured and an aliquot of each culture was taken and diluted to 5 × 10 5 CFU/ml using TSB.These cultures were divided into aliquots and spiked with GT in MeOH (0, 1.875, 3.75 and 7.5 µM) and further divided into aliquots for each time point in falcon tubes.After incubation (37 °C; 170 rpm), culture growth was analysed at 0, 2, 4, 8, and 24 h whereby at each time point the cultures were serially diluted in TSB and streaked on TSB agar.The plates were grown (37 °C; Static) and counted after 24 h.

Determination of DTG and TPEN Zn pKd
This methodology was adapted from that of Kocyła et al., with the 10 h incubation being done in plastic cuvettes instead of glass 60 .

Assessment of DTG and related thiol compound reductant activity by Siderotec™ assays
The Siderotec™ total and Siderotec-HiSens™ tests were performed as per manufacturer's instructions (Accuplex Diagnostics Limited, Ireland).In brief, these tests deploy chromophoric and fluorimetric compounds, respectively, to detect Fe 3+ chelator or reductant activity 61 , Supplemental information and Supplementary Figs. 6 and 7.

Quantitative proteomic analysis of E. faecalis response to GT exposure
Three biological replicate E. faecalis cultures were grown overnight in TSB.These cultures were then used to inoculate warm TSB to an OD600 nm 0.05 and grown (37°C; 170 rpm) until log phase (OD600 nm 0.3).At this stage, each culture was divided into two equal aliquots, one of each was spiked with GT (5 µg/ml final) while the other was spiked with the same volume of MeOH (solvent control).The cultures were returned to the incubator (37°C; 170 rpm) with aliquots taken at T = 30, 60 and 180 min post spiking.Cell pellets were collected and stored at -20°C until required.Pellets were prepared using a modified FASP protocol 62 as follows: pellets were lysed via sonication whereby 25 mg bacteria was resuspended in 150 μl of 1% (w/v) SDS/500 mM ammonium bicarbonate pH 8.3.Lysed samples were heated at 95°C for 5 min.The samples were then centrifuged at 16,000 g for 5 min, supernatants were collected, and protein content quantified using Qubit protein assay.A 20 µg protein sample was taken from each and transferred to 30 kDa molecular weight cut-off (MWCO) centrifugal filters (Sartorius, cat no.VN01H22).Each sample was brought up to 200 µl with 8 M urea, 500 mM ammonium bicarbonate.The samples were vortexed then centrifuged at 14,000 g for 20 min.500 mM ammonium bicarbonate, 8 M urea pH 8.3 (200 µl) was added to each filter.The samples were vortexed, then centrifuged at 14,000 g for 20 min and 100 μl 5 mM TCEP in 8M urea, 500 mM ammonium bicarbonate pH 8.3 was added to each sample filter.The samples were vortexed and incubated at room temperature for 20 min.500 mM IAA (3 µl) was added to each sample (15 mM final) and samples were vortexed and incubated in the dark at room temperature for 20 min.The samples were centrifuged at 14,000 g for 20 min.100 μl of 8 M urea, 500 mM ammonium bicarbonate pH 8.3 was added to each filter.The samples were vortexed then centrifuged at 14,000 g for 20 min.Another 100 μl of 8 M urea, 500 mM ammonium bicarbonate was added to each filter.The samples were vortexed then centrifuged at 14,000

Figure 4 .
Figure 4. DTG ejects specific metal cations (M 2+ ) from 4-(2-Pyridylazo)resorcinol (PAR).DTG removes Zn from PAR:Zn.(A) The effect of increasing DTG concentration on the PAR:Zn complex (495 nm).As the concentration of DTG increases the 495 nm peak decreases accompanied by an increased peak at 410 nm representing unbound PAR.(B) The metal chelator TPEN requires a lower concentration to remove the same amount of metal.(C) DTG removes Cu from PAR:Cu.The effect of increasing DTG concentration on the PAR:Cu complex (513 nm).Interestingly, DTG was able to remove Cu from PAR at lower concentrations than when removing Zn. (D) When this is compared to the efficacy of the metal chelator TPEN there is little difference between the two.

Figure 5 .
Figure 5. DTG specifically reacts with colorimetric and fluorescent iron chelates, possibly via Fe 3+ reduction.DTG specifically removes Fe 3+ from colorimetric or fluorescent Siderotec™ reagents.(A) The impact of gliotoxin, DTG, and TCEP on the colorimetric Fe 3+ assay.The control (purple) indicates the signal at total Fe 3+ removal from the chromophore.DTG (red) is the only test compound which also removes Fe 3+ .(B) The impact of gliotoxin, DTG, and TCEP on the fluorimetric Siderotec HiSens assay.The control (purple) indicates the signal due to Fe 3+ removal from the fluorophore.DTG (red) is the only test compound which also removes Fe 3+ .(C) Temporal effect of DTG on the high sensitivity assay where individual samples were normalized to a percentage relative to the control sample (100%) at each timepoint.Peak fluorescence occurs at T60 min (41% of Fe 3+ removed) which then begins to drop slightly by T120 min.(D) Neither gliotoxin or (E) TCEP affect the fluorometric Siderotec HiSens assay.The control (blue) indicates the signal at total Fe 3+ removal from the fluorophore.

Figure 6 .
Figure 6.Mass spectrometric confirmation of DTG:metal ion chelates.(A) Identification of DTG:Zn chelate structures detected by negative mode mass spectrometry.Structures are shown with Cl -ion and the relevant Zn isotope.(B) The DTG:Cu chelate structures detected by negative mode mass spectrometry.Structures are shown with Cl -ion and the relevant Cu isotope.(C) The DTG:Fe chelate structures detected by negative mode mass spectrometry.Predicted structures are shown with the two DTG molecular structures and the relevant Fe isotope.

Table 2 .
Proteins which are increased in abundance in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 30 min in the log phase (0.3-0.4 OD600).

Table 12 .
Proteins which are decreased in abundance in E. faecalis when grown in the presence of gliotoxin (5 µg/ml), compared to identical cultures grown without gliotoxin (5 µg/ml).Grown using tryptic soy broth (TSB) media for 180 min in the log phase (0.3-0.4 OD600).camera attached to a microscope (Olympus SZX16 microscope with Olympus SDF PLAPO 2XPFC camera attachment) using cellSens Standard software.The wells were homogenised using a multichannel pipette OD600 nm determined using a BioTek Synergy HT 96-well plate reader.