Alpha-1 antitrypsin inhibits Clostridium botulinum C2 toxin, Corynebacterium diphtheriae diphtheria toxin and B. anthracis fusion toxin

The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α1-antitrypsin (α1AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α1AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α1AT. Since α1AT is commercially available as a drug for treatment of the α1AT deficiency, it could be repurposed for treatment of toxin-mediated diseases.


Results α 1 AT inhibits intoxication of HeLa cells with C2 toxin
After identifying α 1 AT as an inhibitor of PT, we explored whether this protective effect could be extended to other AB-type toxins as well and tested C2 toxin, the prototype of clostridial binary toxins (Fig. 1).Since C2 toxin exhibits its enzymatic activity on the key component of the cytoskeleton, G-actin, by ADP-ribosylation, the cytoskeleton collapses which leads to cell rounding, and cell death.The toxin-caused cell rounding and change of morphology was investigated as a specific endpoint for C2 intoxication of HeLa cells.When HeLa cells were intoxicated with C2 toxin in the presence of different concentrations of α 1 AT, a concentration-dependent decrease of cell rounding was observed, indicating inhibition of C2 intoxication by α 1 AT.This effect was more pronounced, when C2 toxin was preincubated with α 1 AT for 15 min at room temperature before addition to HeLa cells (Supplementary Fig. 1).To unravel the mechanism of inhibition by α 1 AT on C2 intoxication, experiments were conducted to investigate the steps of C2 toxin uptake and mode of action in an isolated manner.

α 1 AT inhibits C2 toxin mediated destruction of actin cytoskeleton in a concentration-dependent manner
To further confirm that α 1 AT inhibits C2 toxin intoxication, fluorescence microscopy experiments were performed analyzing the actin cytoskeleton.Therefore, HeLa cells were intoxicated with C2 toxin for 4 h in the presence or absence of different concentrations of α 1 AT, while F-actin was stained (Fig. 2).Untreated control cells were flat and adherent with an organized cytoskeleton and strong signal for F-actin.In contrast, cells treated with C2 toxin showed dot like signals for F-actin, indicating ADP-ribosylation of G-actin with the resulting destruction of F-actin.At the same time the cells exhibited a round appearance further indicating intoxication.
www.nature.com/scientificreports/When cells were treated with increasing concentrations of α 1 AT, the signal for F-actin is comparable with the signal for F-actin in untreated control cells.This protective effect was concentration-dependent with 50 µM  (a, b) Schematic representation of experimental setup for the cell morphology assay.C2 toxin and α 1 AT were either preincubated for 15 min before addition to cells (a) or added simultaneously (b).Then, cells were incubated for 7 h at 37 °C, and pictures were taken every hour using the light microscope (LM).(c-e) Different concentrations of α 1 AT or the respective amount of its solvent (H 2 O) were preincubated for 15 min at RT with C2 toxin (C2 toxin = C2I/ C2IIa: 100/200 ng/ml) in FCS-free medium before addition to HeLa cells (c, d) or added directly (e).The cells were incubated for 7 h at 37 °C, and pictures were taken every hour.Rounded cells are given as percent of the total cell count, mean + /− SEM (at least n = 6 and at most n = 9 values from three independent experiments).(c) Representative pictures are shown for a representative experiment where C2 toxin was preincubated for 15 min with different concentrations of α 1 AT before addition to HeLa cells.and 100 µM α 1 AT showing the strongest effect.When HeLa cells were only treated with 100 µM α 1 AT or when cells were treated with 100 µM α 1 AT but not permeabilized, the signal for membrane-permeant F-actin was not altered (Supplementary Fig. 2).

α 1 AT inhibits the enzyme activity of C2I
Since the modification of actin was inhibited in α 1 AT treated C2 intoxicated HeLa cells, the effect of α 1 AT on the enzyme activity of C2I in vitro was investigated.Here, cell lysate from HeLa cells was used as source for G-actin.As such, C2I, different concentrations of α 1 AT, cell lysate, and biotin-labeled NAD + were incubated together (Fig. 3, Supplementary Fig. 7).G-Actin which was ADP-ribosylated and biotin-labeled via the incubation with C2I and biotin-labeled NAD + , was detected via Western Blot.Interestingly, α 1 AT inhibited the enzyme activity

α 1 AT inhibits binding of labeled C2 to HeLa cells
To further unravel the inhibition mechanism of α 1 AT on C2 intoxication, the binding of C2 toxin to cells was analyzed.Therefore, a flow cytometry based binding assay with fluorescently labeled C2I and C2IIa was performed.
Here, the labeled toxin components were preincubated with 100 µM α 1 AT for 15 min at room temperature.The approaches were subsequently added to HeLa cells and incubated for further 15 min at 4 °C to enable toxin binding but not internalization.As such, a significantly reduced fluorescence intensity was observed in the presence of α 1 AT, regardless of whether the enzyme component C2I or the binding component C2IIa was fluorescently labeled and detected by flow cytometry (Fig. 4).

α 1 AT reduces detectable C2IIa signal in HeLa cells and inhibits binding of C2 toxin to HeLa cells in a concentration-dependent manner
Furthermore, the inhibition of C2 toxin cell binding by α 1 AT was confirmed by monitoring the cell morphology.Therefore, HeLa cells were incubated with α 1 AT and C2 toxin at 4 °C.After medium exchange, cells were transferred to 37 °C, and cell morphology was monitored (Fig. 5a,b).By this, only C2 toxin that has bound to cells during the incubation at 4 °C leads to intoxication and therefore cell rounding.Here, 100 µM α 1 AT almost completely inhibited C2 intoxication by inhibiting C2 binding to HeLa cells.Moreover, binding experiments based on fluorescence microscopy were conducted using labeled C2IIa.This analysis demonstrated as well that C2 toxin binding is reduced by α 1 AT (Fig. 5c).To investigate whether inhibition of C2 binding by α 1 AT is directed due to C2-α 1 AT precipitate formation or cell membrane-α 1 AT interaction a precipitation analysis (Supplementary Fig. 3, Supplementary Fig. 8) and an assay monitoring cell rounding were conducted (Supplementary Fig. 4) respectively.For the precipitation analysis, TcdB and α 1 AT or α-Defensin 6 as a positive control or C2 toxin with different concentrations of α 1 AT were incubated for 30 min at 37 °C.After that the samples were centrifuged and supernatant and pellet fraction were analyzed using Western Blot.Here, TcdB and C2II/C2IIa were detected.As shown in Supplementary Fig. 3b TcdB does not precipitate with α 1 AT, since most of the TcdB signal is observed in the supernatant fraction.As previously reported 50 , TcdB and α-Defensin 6 form precipitates as the majority of the signal is detected in the pellet fraction.For all the approaches with C2 and α 1 AT in Supplementary Fig. 3c, most of the signal is observed in the supernatant fraction.As such, no concentration-dependent precipitate formation was detected.To test whether α 1 AT inhibits C2 binding by blocking binding sites on the cell surface, a cell morphology assay was conducted where α 1 AT binding to cells was allowed for 40 min at 4 °C.After that, a washing step was performed to remove unbound α 1 AT and the cells were intoxicated with C2 toxin at 37 °C.Then cell rounding was monitored.As shown in Supplementary Fig. 4, pre-incubation of cells with α 1 AT could not inhibit intoxication of cells with C2 toxin.This suggests that presence of α 1 AT during C2 intoxication is required for sufficient toxin inhibition.
Since C2 binding was inhibited by α 1 AT, a fluorescence microscopy experiment was conducted to investigate the endocytosis of C2 in presence of α 1 AT.Therefore, once again fluorescently labeled C2IIa was used.For the investigation of endocytosis, cells were treated with C2 toxin and different concentrations of α 1 AT for 40 min at 37 °C.As such, due to inhibition of C2 binding to HeLa cells also less endocytosed C2 toxin was observed (Fig. 6).

α 1 AT inhibits other bacterial AB-type toxins in a concentration-dependent manner
To further investigate the inhibitory capacity of α 1 AT, other bacterial AB-type toxins were tested using an assay monitoring the cell morphology (Fig. 7, Supplementary Fig. 5, and Table 1).Moreover, we recently have published that α 1 AT inhibits binding of PT from Bordetella pertussis due to direct interaction of the PT binding subunit with α 1 AT.
Diphtheria toxin (DT) is a single-chain ADP-ribosylating toxin that is taken up into cells by receptor-mediated endocytosis and translocates its enzyme domain from early endosomes into the cytosol to modify elongation factor 2. In HeLa cells, intoxication with DT leads to cell rounding 51 .DT requires proteolytic activation by the membrane-anchored protease furin to enter cells.However, DT as well as proteolytically activated DT, nicked DT (nDT), were both inhibited by α 1 AT in a comparable manner (Fig. 7b,c).This indicates that inhibition of DT by α 1 AT might also be independent of α 1 AT's ability to inhibit serine proteases or other proteases such as furin.Flow cytometry analysis revealed that α 1 AT did not hinder the binding of an enzymatically inactive, eGFP-fused Subsequently, the cells were washed, fixed, permeabilized (as indicated), and quenching was performed.Blocking was performed, and the cells were incubated with a primary antibody for α 1 AT (purple).Primary antibody was detected via a fluorescently labeled secondary antibody, F-actin was stained using sir-actin (red), and nuclei were stained using Hoechst (blue).
Representative images are shown from three independent experiments (n = 3).www.nature.com/scientificreports/DT variant (His-eGFP-CRM197) to cells (Supplementary Fig. 6), suggesting a distinct inhibition mechanism compared to that of C2 toxin and PT.Further single-chain AB-toxins translocating their enzyme domains from endosomes to the cytosol are C. difficile toxins TcdA and TcdB.In contrast to DT and PT, TcdA and TcdB have glucosyltransferase activity targeting small Rho GTPases.This leads also to cell rounding.No inhibition of α 1 AT on intoxication with TcdA or TcdB was observed (Fig. 7d,e).
Next to single-chain and AB 5 -type toxins, binary toxins show a distinct structure harboring their A-and B-domains on two separate proteins, then called components.Clostridium (C.) botulinum C2 toxin and C. difficile CDT are binary toxins that both translocate their enzyme components from early endosomes and ADPribosylate G-actin in the cytosol.This also leads to rounding of adherent cells.Interestingly, α 1 AT protected cells from intoxication by C2 toxin but not by CDT (Fig. 7f).
These results together with the finding that α 1 AT inhibits the binding of PT to cells, prompted us to test different combinations of binding/transport and enzyme domains of toxins.First, we tested whether the transport of the enzyme domain of TcdB, His_TcdB-GTD via the transport component of C2 toxin, C2IIa or the transport component of anthrax toxin, protective antigen (PA63), is inhibited by α 1 AT.It was recently shown that C2IIa not only can transport its enzyme component C2I but also His-tagged enzymes into the cytosol of cells 52 .Moreover, transport of His-tagged enzymes was also shown for the transport component of anthrax toxin, PA63 53,54 .Comparable to DT, PA83 of anthrax toxin undergoes proteolytic cleavage by furin, resulting in PA63, which is required for its toxic activity 55 .Figure 7g reveals that α 1 AT inhibits intoxication of cells by the combination of C2IIa and His_TcdB-GTD in a concentration-dependent manner.Figure 7h show inhibition of intoxication with the combination of PA63 and His_TcdB-GTD by α 1 AT.Next, we tested whether the transport of the fusion protein LF N -DTA, where LF N mediates the uptake of the enzyme domain DTA of DT into the cytosol, via the transport component of anthrax toxin, PA63, is inhibited by α 1 AT.LF N is the N-terminal part of the enzyme component of anthrax lethal toxin which mediates the interaction with PA63.DTA was fused to LF N to obtain a robust morphological readout (cell rounding).Results in Fig. 7i also show only inhibition by α 1 AT if cells were intoxicated with the combination of PA63 and LF N -DTA.Taken together, these results indicate the binding subunit of the toxin most likely determines whether cells are protected from intoxication by α 1 AT.
Taken together, the results underscore that α 1 AT exerts inhibitory effects not only on C2 toxin and PT but also on diverse bacterial AB-type toxins.Notably, the inhibition of toxin binding emerges as a pivotal mechanism, complemented by the modulation of enzyme activity.This multifaceted inhibition suggests the promise of α 1 AT as a therapeutic agent against a spectrum of toxin-mediated diseases, proposing a versatile strategy for treating multiple pathologies rather than targeting individual afflictions exclusively.

Discussion
In this study, we explored the inhibitory effects of α 1 AT on the C2 toxin produced by Clostridium botulinum, diphtheria toxin, and an anthrax fusion toxin.Our findings demonstrate that α 1 AT, a serine protease inhibitor identified from a hemofiltrate library screen for inhibitors of PT from Bordetella pertussis, is a multitoxin inhibitor.α 1 AT is essential for regulating protease activity and controlling inflammation.It protects lung tissue from damage caused by enzymes like neutrophil elastase, released by immune cells 42 .In healthy individuals, α 1 AT plasma levels range from 0.9 to 2 mg/ml (approximately 17-38 μM).During acute inflammation, these levels can increase four to five times, highlighting α 1 AT's role as an acute phase protein in preventing inflammation-induced Table 1.Summary on tested toxins for α 1 AT.Seven toxins were tested for inhibition of α 1 AT.Summarized are the bacterial origin, toxin structure and function, the intoxication route (short trip: enzyme domains translocate from endosomes into the cytosol; long trip: retrograde trafficking from toxin through endosomes, Golgi apparatus, and ER, enzyme domains translocate from the ER into the cytosol), their molecular target, and the mechanism of toxin inhibition by α 1 AT.www.nature.com/scientificreports/tissue damage 40 .Interestingly, we have observed that in Bordetella Pertussis infected mice mRNA levels of the murine genes of α 1 AT, Serpin1a-e, were downregulated (Lietz et al., under revision).This suggests that during bacterial infection with Bordetella Pertussis α 1 AT levels might be reduced, ameliorating lung tissue damage, and toxin function due to protease imbalance in favor of neutrophil elastase.In the lungs, physiological α 1 AT concentrations range from 10 to 40 μM in alveolar interstitial fluid and about 2-5 μM in alveolar extracellular lining fluid 43,47,56 .Those concentrations have shown to be protective in our cell-based experiments but if α 1 AT levels are reduced protection against bacterial toxins might be lost.Analyzing serum levels of α 1 AT in infected patients would give further valuable insights.α 1 AT products like Prolastin, derived from purified donated blood, have been used to treat genetic α 1 AT deficiency for decades 44 .These products are typically given intravenously to address chronic tissue degradation in the lower respiratory tract due to the deficiency.Alternatively, α 1 AT can be administered via inhalation, enabling much higher doses than standard treatments 41 .Doses as high as 250 mg/kg have shown a fivefold increase in serpin concentration within the lung's epithelial lining fluid without causing adverse effects 47 .
Our study found that α 1 AT protects cells from intoxication with C2 toxin, DT, and anthrax fusion toxin starting at concentrations around 10-30 μM, which are within the physiological range.The protective effect was more pronounced at higher concentrations, with the most significant impact observed at concentrations up to 100 μM.Interestingly, comparable concentrations have been previously found to inhibit PT (Lietz et al., under revision).The identification of α 1 AT as an inhibitor of multiple clinically relevant toxins, including C2 toxin, DT, anthrax toxin, and PT highlights its potential for broad-spectrum anti-toxin therapy.The repurposing of α 1 AT could provide a rapid and effective treatment option for various toxin-mediated illnesses, leveraging its established clinical use and safety profile.
Specifically, α 1 AT inhibits the intoxication of cells by C2 toxin through two distinct mechanisms: inhibition of C2 toxin binding to cells and inhibition of the enzyme activity of the C2I component.This dual mechanism of action enhances its efficacy as an inhibitor.Inhibition of binding is most likely directed via the interaction of C2 toxin and α 1 AT, since pre-incubation of cells with α 1 AT was not sufficient for inhibition of C2 intoxication.However, no precipitate formation was observed, suggesting that C2 toxin and α 1 AT do not from aggregates.Moreover, our study found that α 1 AT inhibits both C2 toxin and anthrax fusion toxin, as well as the enzyme domain His_TcdB-GTD transported by PA63, the binding component of anthrax toxin.Moreover, also DT and nicked DT were inhibited by α 1 AT.As a common feature many bacterial AB-type toxins require activation via furin cleavage.Taken our results together, inhibition of furin by α 1 AT might be part of the inhibitory mechanism of α 1 AT, but inhibition of these toxins does not solely rely on inhibition of furin, since also activated toxins (nicked DT and PA63) and toxins that are not dependent on furin cleavage are inhibited.However, others have shown that an engineered α 1 AT variant, α 1 AT Portland, designed as a furin inhibitor can be employed as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A and is able to form SDS-and heat-stable serpin/proteinase complexes 57,58 .As such, furin inhibition by α 1 AT plays a neglectable role in the context of our study.Moreover, previous results (Lietz et al., under revision) have shown that another member of the serpin-family, antithrombin, was not able to inhibit PT, showing that protease inhibition activity is not required for toxin inhibition and that basis of inhibitory capacity of α 1 AT probably lies within unique characteristics of structure or amino acid composition.
In contrast, α 1 AT did not inhibit TcdA, TcdB or CDT toxin.All five toxins use different cell surface receptors 7,27,59,60 .C2 toxin, anthrax toxin and CDT belong to the group of binary toxins and share between approximately 30-40% sequence homology in their activated B-components 61 .However, it was recently shown that the crystal structure of CDTb differs from the PA structure and an additional receptor-binding domain was discovered in CDTb, which is absent in the protective antigen 62 .This finding opens up new avenues for research into the design of inhibitors that can target structurally similar toxins, potentially broadening the scope of α 1 AT's therapeutic applications.Further research is needed to fully understand these mechanisms and to explore the potential for α 1 AT to inhibit other similar toxins.
Anthrax toxin, produced by Bacillus anthracis, remains a significant concern due to its potential use as a bioterrorism agent and its role in zoonotic infections 37 .The toxin comprises three components: protective antigen (PA; PA63, proteolytically activated), lethal factor (LF), and edema factor (EF) 27 .PA binds to host cell receptors and facilitates the entry of LF and EF into cells, leading to lethal and edema responses, respectively.Here, we investigated the anthrax fusion toxin PA63 + LF N DTA consisting of the N-terminal part of LF (LF N ) that facilitates the interaction with PA63 and the enzyme domain of diphtheria toxin (DTA) 32 .This allows a morphological readout on cultivated HeLa cells due to DTA-induced cell rounding while investigating the effect of α 1 AT on PA63 and LF N .α 1 AT inhibited intoxication with PA + LF N DTA most likely due to inhibition of PA63-binding to cells.This is supported by the finding that intoxication with His_TcdB-GTD + PA63 is also inhibited by α 1 AT although intoxication with the wildtype TcdB with its original receptor domain is not affected by α 1 AT.The historical impact of anthrax, its high mortality rate when inhaled, and the ease of spore dissemination underscore the need for effective countermeasures.The ability of α 1 AT to inhibit anthrax toxin adds a potential dimension to its therapeutic potential, offering a promising defense against both natural outbreaks and bioterrorism threats.Developing α 1 AT as an inhibitor could provide a rapid, effective response to anthrax exposure, thereby enhancing public health security.
Although a widespread vaccination against diphtheria is available, outbreaks in Bangladesh, Haiti, and South Africa have been reported recently 25,26 .Additionally, the number of cases worldwide has been increasing in recent years, leading to C. diphtheriae being considered a re-emerging pathogen 26 .Treatment strategies for diphtheria could benefit from incorporating α 1 AT, particularly since high concentrations can be achieved through inhalation.This method aligns well with the disease's pathology, as DT is primarily released in the upper respiratory tract, where it causes severe symptoms and airway obstruction 19,63  www.nature.com/scientificreports/ While our study provides compelling evidence for the inhibitory effects of α 1 AT on multiple toxins, it is limited by its in vitro nature using cell culture experimentation.Future studies should include in vivo experiments to confirm these findings and assess the therapeutic potential of α 1 AT in animal models.Additionally, exploring the structure-activity relationship of α 1 AT and its interactions with various toxins could lead to the development of optimized inhibitors.In conclusion, α 1 AT shows promise as a multitoxin inhibitor with potential applications in treating toxin-mediated diseases.Its established use in treating α 1 AT deficiency, coupled with its broad-spectrum inhibitory effects, supports its repurposing as an anti-toxin therapeutic.Further research and in vivo studies are necessary to fully realize its potential in clinical settings.

Compounds and reagents
The native toxins TcdA and TcdB from C. difficile VPI 10,463 were expressed and purified as described elsewhere 64 .C2I and C2IIa were purified according to 65 .His_TcdB-GTD was purified as described earlier 52 .DT was purchased from Calbiochem/Merck KGaA (Bad Soden/Darmstadt, Germany).DT was activated in vitro by trypsin digestion as described before 51 , resulting in nicked DT (nDT).His-eGFP-CRM197 was expressed and purified as described before 66 , as well as CDTa and CDTb 67 and PA63 52 .The fusion toxin LF N -DTA was expressed and purified as described earlier 32 .As α 1 AT source the commercially available drug Prolastin ® purchased from Grifols (Frankfurt am Main, Germany) was used.The peptide α-defensin-6 was purchased from PeptaNova (Sandhausen, Germany).

Cell lines
Unless mentioned differently, the materials for the cultivation of all cell lines were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA).The used cell lines included Vero cells (African green monkey kidney cells; DSMZ, Braunschweig, Germany), and HeLa cells (cervical carcinoma cells; DSMZ, Braunschweig, Germany).Vero cells and HeLa cells were cultivated in MEM, while MEM was supplemented with 10% FCS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 100 U/ml penicillin and 100 g/ml streptomycin.Cells were cultivated under humidified conditions at 37 °C with 5% CO 2 and trypsinized and reseeded every two to three days for at most 25 times.For intoxication experiments, cells were seeded in culture dishes one or two days before and treated in FCS-free media with toxins and the respective compounds.

Cell morphology assays
For the analysis of cell morphological changes, cells were seeded in 96-well plates one or two days prior to treatment with toxins, α 1 AT, and water (solvent control).Before the addition of the components to cells, α 1 AT or water (solvent control) was preincubated with the respective toxin for 15 min at room temperature in FCS-free medium.The morphology of cells was documented using light microscopy every hour for at least 6 h using 20 × magnification a Leica DMi1 microscope connected to a Leica MC170 HD camera (both Leica Microsystems GmbH, Wetzlar, Germany).Rounded cells were counted from complete pictures taken, using the online software Neuralab.

Enzyme activity assay of C2I from cell lysates
For the analysis of the in vitro enzyme activity of C2I, cell lysate from HeLa cells was generated.As such, HeLa cells were seeded in 10 cm culture dishes and grown for two to three days.After that, the cells were washed, and frozen for lysis.Next, ADP-ribosylation buffer 20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2, 1:50 freshly added cOmplete ™ (Roche); pH 7.5) was added, and the cell lysate was collected in tubes.The cell lysate was centrifuged at 10,000 × g for 1 min, the supernatant was transferred into a new tube, and the protein concentration was measured using the Nanodrop.Different concentrations of α 1 AT, water (solvent control), and 30 µg HeLa cell lysate were mixed in ADP-ribosylation buffer with a total reaction volume of 20 µl.Subsequently, C2I (20 fmol = 0.001 µM) and biotin-labeled NAD + (1 µM; R&D Systems) were added for labeling of the ADP-ribosylation of actin during the 30 min incubation at 37 °C.After that, the samples were subjected to gel electrophoresis and Western blotting.The ADP-ribosylated actin by C2I was detected using streptavidinperoxidase (Strep-POD, Sigma-Aldrich, Merck, Darmstadt, Germany).Hsp90 (primary antibody from Santa Cruz Biotechnology, Dallas, TX, USA) served as loading control, and the signal quantification was performed using the ImageJ software v.1.52.a (NIH).

Gel electrophoresis and western blotting
After the samples were prepared, Laemmli buffer (0.3 M Tris-HCl, 10% SDS, 37.5% glycerol, 0.4 mM bromophenol blue, 100 mM DTT) was added, and the samples were heat-denatured at 95 °C for 10 min.For protein separation via gel electrophoresis, 12.5% acrylamide gels were used.The transfer of proteins from the gels onto nitrocellulose membranes was performed by semi-dry Western blotting and controlled by staining the membranes with Ponceau-S-staining (AppliChem GmbH, Darmstadt, Germany).Then, the membranes were blocked in 5% skim milk powder in PBS-T (PBS containing 0.1% Tween 20) for at least 30 min at room temperature, followed by washing steps in PBS-T.Next, incubation with primary and secondary antibodies in PBS-T separated by washing steps was performed.After the final washing steps, signals were detected using Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA) and X-ray films (AGFA Health Care, Mortsel, Belgium) or the iBright 1500 system (Thermo Fisher Scientific).www.nature.com/scientificreports/

Binding analysis using flow cytometry
For the binding analysis based on flow cytometry, C2I was labeled with DyLight ® 405 NHS Ester (Thermo Fisher Scientific, Rockford, IL, USA) and C2IIa was labeled with DyLight ® 488 NHS Ester (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's protocol using the Zeba ™ Spin Desalting Columns (7 K MWCO, Thermo Fisher Scientific, Waltham, MA, USA) to remove excess dye.After that, cells were grown in a culture dish, detached using 25 mM EDTA in PBS, and resuspended in PBS.488-labeled C2IIa and 405-labeled C2I or His_eGFP_CRM197, α 1 AT, and water (solvent control) were preincubated for 15 min at room temperature or added directly to cells (1 or 2 × 10 6 in 0.2 mL per sample) for 15 min at 4 °C to enable binding of C2 or His_eGFP_CRM197 to cells but no internalization.The samples were washed by centrifugation, and the cell fluorescence was measured at an excitation wavelength of 488 nm or 405 nm using the BD FACS Celesta™ flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and the BD FACSDiva ™ software 8.0.1.1.Cell gating and data analysis was performed using Flowing Software v2.5.1.(Turku Centre of Biotechnology, Finland).

Cellular binding and uptake analysis using fluorescence microscopy
For all immunostaining and fluorescence microscopy experiments, cells were seeded and grown for one day in 18-well µ-slides (ibidi GmbH, Gräfelfing, Germany).Subsequently, the cells were treated with α 1 AT or water (solvent control) and were intoxicated with C2 toxin (C2IIa-488 or C2IIa and C2I) in FCS-free medium for the uptake analysis for 4 h at 37 °C, for the endocytose analysis for 40 min at 37 °C or for the binding analysis for 40 min at 4 °C.Approaches including labeled C2IIa were centrifuged before addition to cells.After that, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized using 0.4% (v/v) Triton X-100 in PBS for 5 min if required, and quenching was performed for 2 min in glycine (100 mM in PBS).After that, the cells were blocked for 1 h at 37 °C in PBS-T (PBS containing 0.1% Tween 20) containing 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA) and 1% BSA.The incubation with the primary antibody for α 1 AT (Alpha-1-Antitrypsin antibody, Proteintech, Planegg-Martinsried, Germany) in blocking solution was performed for 1 h at 37 °C.F-actin was stained for 1 h at 37 °C using the membrane-permeant siractin (SiR-actin kit, Spirochrome, Stein am Rhein, Switzerland).The primary antibody for α 1 AT was detected via fluorescently labeled secondary antibody in blocking solution for 1 h at 37 °C, and cell nuclei were stained for 5 min using Hoechst 33,342 (1:10,000, Thermo Fisher Scientific, Waltham, MA, USA).After staining, the slides were examined via microscopy using the BZ-X810 Keyence fluorescence microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany) and BZ-X800Viewer v1.3.0.

In vitro precipitation assay
For the in vitro precipitation analysis, C2 (C2IIa/C2I: 33.2/20 nM) and TcdB (50 ng) and inhibitors, α 1 AT and α-defensin-6 (6 µM) were incubated for 30 min in PBS at 37 °C.After incubation, the samples were centrifuged for 20 min, 14,000 rpm at 4 °C.Subsequently, the supernatant fraction was separated and transferred into a new tube and the pellet was resuspended in PBS.The samples were subjected to gel electrophoresis and Western blotting.C2 was detected using an antiserum against C2II while TcdB was detected using an anti-TcdB antibody (Anti-Clostridium difficile Toxin B antibody, Abcam, Cambridge, UK).

Fig. 1 .
Fig. 1.Effect of α 1 AT on C2-toxin mediated cell rounding of HeLa cells.(a,b) Schematic representation of experimental setup for the cell morphology assay.C2 toxin and α 1 AT were either preincubated for 15 min before addition to cells (a) or added simultaneously (b).Then, cells were incubated for 7 h at 37 °C, and pictures were taken every hour using the light microscope (LM).(c-e) Different concentrations of α 1 AT or the respective amount of its solvent (H 2 O) were preincubated for 15 min at RT with C2 toxin (C2 toxin = C2I/ C2IIa: 100/200 ng/ml) in FCS-free medium before addition to HeLa cells (c, d) or added directly (e).The cells were incubated for 7 h at 37 °C, and pictures were taken every hour.Rounded cells are given as percent of the total cell count, mean + /− SEM (at least n = 6 and at most n = 9 values from three independent experiments).(c) Representative pictures are shown for a representative experiment where C2 toxin was preincubated for 15 min with different concentrations of α 1 AT before addition to HeLa cells.

Fig. 2 .
Fig. 2.Effect of α 1 AT on F-actin during intoxication of HeLa cells with C2 toxin.C2 toxin (C2I/C2IIa: 100/200 ng/ml) and different concentrations α 1 AT or the respective amount of solvent (H 2 O) were added directly to HeLa cells and incubated for 4 h at 37 °C.Cells were left untreated as control (Con).Subsequently, the cells were washed, fixed, permeabilized, and quenching was performed.Blocking was performed and F-actin was stained using sir-actin (red), and nuclei were stained using Hoechst (blue).Representative images are shown from three independent experiments (n = 3).

Fig. 3 .
Fig. 3. Effect of α 1 AT on enzyme activity of C2I in vitro.(a) Schematic representation of experimental setup for the enzyme activity assay.C2I and α 1 AT were directly added to HeLa cell lysate and biotin-NAD + , and incubated for 30 min at 37 °C.(b-d) C2I (20 fmol = 0.001 µM) and different concentrations α 1 AT or the respective amount of solvent (H 2 O) (Con) were added directly to HeLa cell lysate and biotin-NAD + and incubated for 30 min at 37 °C.Cell lysate was left untreated with biotin-NAD + as further control (Lysate).G-Actin which was ADPribosylated and biotin-labeled via the incubation with C2I and biotin-labeled NAD + was detected via Western Blot (WB), whereas Hsp90 or Ponceau-S staining served as control for equal protein loading.The bar graph (b) shows the quantifications of Western Blot signals from nine independent experiments, while (c, d) show blots of representative experiments.The intensity values of the bar graph are given as x-fold of the control (con), normalized to Hsp90 or Ponceau-S staining, mean + /− SEM (at least n = 4 at most n = 16 values from nine independent experiments).(b) Significance was tested using one-way ANOVA followed by Dunnett's multiple comparison test and refers to untreated controls (con) (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant).

Fig. 4 .Fig. 5 .
Fig. 4.Effect of α 1 AT on binding of labeled C2 toxin to HeLa cells.(a-d) 488-labeled C2IIa (800 ng/ml) and 405-labeled C2I (800 ng/ml) and 100 µM of α 1 AT or the respective amount of solvent (H 2 O) were added directly to HeLa cells and incubated for 15 min at 4 °C to enable binding of C2 toxin to cells but no internalization.Cells were left untreated (PBS controls from labeling process) as control.After that, cells were washed by centrifugation and 488-labeled C2IIa (a) and 405-labeled C2I (c) bound to cell surfaces was measured using flow cytometry.Values of median are given as x-fold of the untreated control (Con), mean + /− SEM (n = 9 from three independent experiments).(b, d) Histograms show fluorescence intensity of cells for one representative experiment.(a-d) Significance was tested using one-way ANOVA followed by Dunnett's multiple comparison test and refers to toxin-treated controls (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant).

Fig. 6 .Fig. 7 .
Fig.6.Effect of α 1 AT on endocytosed C2IIa-488 signal in HeLa cells.C2 toxin (C2IIa-488/C2I: 33.2/20 nM) and different concentrations α 1 AT or the respective amount of solvent (H 2 O) were mixed, centrifuged, and supernatants were added directly to HeLa cells and incubated for 30 min at 37 °C.Cells were left untreated as control (Con) or treated with respective amounts of PBS-488 (control from toxin labeling) and solvent (H 2 O) (Con + PBS-488).Subsequently, the cells were washed, fixed, permeabilized (as indicated), and quenching was performed.Blocking was performed, and the cells were incubated with a primary antibody for α 1 AT (purple).Primary antibody was detected via a fluorescently labeled secondary antibody, F-actin was stained using siractin (red), and nuclei were stained using Hoechst (blue).Labelled C2IIa is shown in green.Representative images are shown from three independent experiments (n = 3).
All experiments were performed independently from each other at least three times.The number of replicates (n) for experiments or tested conditions is given in the figure legends.Moreover, representative results are shown in the figures and if not stated otherwise in the figure legends, the statistical analysis performed was a one-way ANOVA in combination with Dunnett's multiple comparison test using GraphPad Prism Version 9 (GraphPad Software Inc., San Diego, CA, USA).The obtained p values are depicted as follows: ns = not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. .