NEDD8 is a ubiquitin-like molecule that is conjugated to lysine residues of protein substrates in a process known as neddylation, which affects their stability, degradation, and interactions. It was recently found that the neddylation inhibitor MLN4924 prevents endocytic uptake of herpes simplex virus (HSV) into target cells, implicating a broader role for neddylation in viral entry and infection. Because AB-type bacterial toxins share similar cell entry mechanisms with viruses, we studied the impact of neddylation in the cellular internalization of a prototypical, well-established model of bacterial AB-type toxins, namely the diphtheria toxin (DT). We found that pretreatment of HeLa cells with MLN4924 reduced morphological changes and protein biosynthesis inhibition induced by DT. Arctigenin, another neddylation inhibitor acting at a later step of the neddylation cascade, was also capable of reducing DT intoxication in HeLa cells. In combination, both neddylation inhibitors synergistically increased their inhibitory potential against DT. Further mechanistic studies with MLN4924 revealed that the neddylation inhibitor neither interfered with cell surface binding, proteolytic activation, enzymatic activity, and membrane translocation of DT, nor with endosomal acidification in HeLa cells. Finally, we could demonstrate that pharmacological inhibition of neddylation also affects further bacterial protein toxins, namely single-chain toxin Clostridioides difficile TcdB and binary toxin Clostridium botulinum C2. In conclusion, our study is the first to support a connection between neddylation and the cellular uptake of bacterial AB-type toxins, thereby establishing a foundation for future investigations into these molecular mechanisms and for the development of anti-toxin strategies.IMPORTANCENeddylation, a ubiquitin-like protein modification, is emerging as a critical regulator of viral entry into cells. Here, we demonstrate for the first time that pharmacological inhibition of neddylation impairs the intoxication of target cells with diverse bacterial AB-type toxins. Mechanistic studies suggest that neddylation most likely reduces toxin internalization, thus supporting the connection between neddylation and endocytic processes. Our study offers new opportunities to explore anti-toxin interventions and highlights the use of bacterial toxins as molecular probes to further study the role of neddylation in endocytic trafficking.

Here, we report the development and comprehensive characterization of copper-chitosan (Cu/CS) antimicrobial films, investigated using a combination of spectroscopic techniques and atomic force microscopy (AFM), to elucidate their chemical composition, Cu2+ ionic release behavior, and physicochemical properties. Particular emphasis was placed on correlating the chemical and surface properties of the films with their antimicrobial performance, to relate the effects of Cu2+ release and physicochemical properties of the films to the antimicrobial action. AFM was employed to analyze the morphology, surface roughness, and integrity of Escherichia coli bacterial cells incubated on Cu/CS films for short (2 h) and prolonged (20 h) exposure times. Time-dependent morphological changes were observed in bacteria in contact with the Cu/CS surfaces, indicating pronounced cellular stress and progressive loss of viability over exposure time. These changes included a transition from the typical rod-like morphology of healthy E. coli to coccoid-shaped cells, the formation of membrane bleb-like protrusions, increased cell clustering, and severe membrane disruption ending in cell lysis. The observed variations in bacterial morphology and surface characteristics provided a direct proof of damage to the bacterial envelope and reduced cellular viability. These findings strongly suggest a combined antimicrobial effect arising from the simultaneous action of Cu2+ ion release (220 ppb in 24 h) and the intrinsic bioactivity of the CS matrix. The results highlight the potential of Cu/CS films as effective antimicrobial materials and support their further development for applications in antimicrobial coatings, packaging, and biomedical surfaces.

In addition to fibrinogens canonical function in hemostasis, alternate roles in innate immunity have emerged. However, interactions with bacterial protein toxins, the main virulence factors of many medically relevant bacteria and causative agents of life-threatening diseases, have not been described so far. Here, we identified human fibrinogen as an inhibitor of the enterotoxic Clostridium (C.) botulinum C2 toxin. Our results indicate that fibrinogen specifically interacts with the binding subunit of C2 toxin in vitro and that N-linked glycans of fibrinogen are crucial for this interaction. This prevents receptor-binding and cellular uptake of the toxin. Related toxins from Bacillus anthracis (lethal toxin), C. perfringens (iota) or Clostridioides difficile (CDT) do not bind to fibrinogen and are therefore not inhibited. Furthermore, C2 toxin had no effect on coagulation of human blood ex vivo. In conclusion, we identified fibrinogen as an inhibitor of a highly potent bacterial protein toxin, highlighting an unexpected role for this blood coagulation factor in innate immunity.

Many highly infectious and severe diseases such as pertussis, diphtheria, anthrax, and Clostridioides difficile infections are caused by bacteria which release symptom-inducing AB-type protein toxins. These diseases are treated using antibiotics or preventive measures including vaccination. However, despite preventive measures, increasing case numbers have been reported in past years. Therefore, novel therapeutic options against toxin-mediated disease are urgently required. Since the toxins are the causative agents of the diseases, the development of pharmacological inhibitors that specifically neutralize individual toxins should be a relevant strategy to further support the current therapies. Huge potential to identify toxin inhibitors lies within the human proteome/peptidome that might contain endogenous toxin inhibitors as yet unknown part of the innate immunity. Screening of human peptide libraries and systematic testing of proteins/peptides with antimicrobial activity led to the identification of proteins/peptides with specific anti-toxin activities. Consequently, defensins, α1-antitrypsin and derived peptides, human serum albumin, and in silico predicted angiogenin-derived peptides were identified as potent inhibitors for bacterial toxins including Clostridioides difficile toxins TcdA, TcdB and CDT, diphtheria, anthrax, and pertussis toxin, and clostridial binary iota and C2 toxins. This review summarizes the current state of identified endogenous proteins/peptides as novel inhibitors for clinically relevant bacterial AB-type toxins.

The life-threatening disease pertussis, also known as whooping cough, is caused by a complex interplay of several virulence factors produced by the bacterium Bordetella (B.) pertussis. These include the AB-type protein toxin pertussis toxin (PT), the main causative agent of pertussis. After infection with B. pertussis, PT is released and binds to its human target cells, which internalize PT. The enzyme subunit of PT is then taken up into the cytosol, where it catalyzes the ADP-ribosylation of the α-subunit of inhibitory GTP-binding proteins from the Gαi type. This ultimately leads to the development of the characteristic clinical symptoms associated with pertussis. Pertussis is a vaccine-preventable but highly infectious respiratory disease, and especially younger children are prone to develop severe pertussis. Despite the vaccination, over the past few years, increasing case numbers have been reported globally. Moreover, treatment options are strongly limited to antibiotics and symptomatic treatment. Therefore, novel therapies against toxin-mediated diseases are urgently required, while AB-type toxins such as PT are promising pharmacological targets to combat these associated diseases. To identify novel pharmacological inhibitors for AB-type toxins, huge potential lies within the human proteome/peptidome. Endogenous protein or peptide inhibitors for bacterial toxins might have evolved as part of the innate immunity and are awaited to be discovered. The scientific community is committed to identify potential candidates through targeted screening or explorative hypothesis-driven approaches. This review summarizes the recent efforts in the identification and characterization of the human body’s own proteins and peptides that inhibit PT. PT-inhibiting peptides were found by unbiased screening of peptide libraries from human hemofiltrate or hypothesis-driven evaluation, and PT-neutralizing mechanisms were discovered in cell-based approaches. The identification of endogenous peptides and proteins, e.g., defensins and α1-antitrypsin, as potent inhibitors of PT paves the way towards the development of novel therapeutic options against pertussis.

Efficient cytosolic delivery remains a major challenge for intracellular protein therapeutics due to endosomal entrapment following cellular uptake. Here, we employ the enzymatically inactive Clostridium botulinum C3E174Q (C3EQ) as an endosomal escape promoter in combination with mesoporous silica nanoparticles (MSNs). C3EQ is selectively adsorbed onto the outer surface of the MSNs through size exclusion. Using the enzymatically active variant C3bot (C3), efficient endosomal escape into the cytosol of J774A.1 macrophages was demonstrated by characteristic Rho-dependent morphological changes. Co-loading C3EQ with the membrane-impermeable peptide NapFab resulted in a more diffuse intracellular peptide distribution than that observed for control nanoparticles, indicating enhanced cytosolic peptide release while the nanoparticles remained compartmentalized. Collectively, these findings demonstrate that surface-adsorbed C3EQ promotes efficient cytosolic delivery of nanoparticle cargo while preserving the intrinsic properties of the nanocarrier, highlighting its potential as a biologically derived platform for macrophage-targeted intracellular drug delivery.

Diphtheria toxin (DT), an AB-type protein exotoxin, is the main virulence factor of Corynebacterium diphtheriae and the causative agent of diphtheria, a life-threatening disease especially in children. DT binds to its receptor heparin-binding EGF-like growth factor on human target cells via its binding subunit DTB, enters cells by receptor-mediated endocytosis and delivers its enzyme subunit DTA into the cytosol. There, DTA catalyzes the ADP-ribosylation of elongation factor 2, which inhibits protein synthesis and leads to cell death. The number of diphtheria cases is increasing worldwide despite routine vaccination against DT in many countries. Although diphtheria mortality is significantly reduced by standard diphtheria antitoxin therapy, there are drawbacks to this therapy and new therapeutic strategies are highly desirable. In this study, we identified the approved anticoagulant drug fondaparinux, the pharmacologically active pentasaccharide sequence within heparin, as potent inhibitor against DT in vitro. Fondaparinux protected eukaryotic cells from intoxication with DT, whereas unfractionated and low molecular weight heparins did not. When DT was applied to cells in the presence of fondaparinux, the ADP-ribosylation of elongation factor 2 was significantly reduced in these cells and their protein synthesis was maintained. By investigating the inhibitory mechanisms of fondaparinux against DT, we found that fondaparinux prevented DT binding to cells and thus DT uptake. As fondaparinux is a licensed drug with well-known toxicity and pharmacokinetic profiles, these findings should provide a starting point for the development of novel pharmacological strategies to treat diphtheria, which is considered a re-emerging disease, also in western countries.

Metastatic breast carcinoma (BC) cells are prone to spreading in the bone microenvironment, leading to a vicious cycle between local osteoclast-mediated osteolysis and tumor progression. Therefore, the targeted pharmacological down-modulation of BC cell proliferation as well as osteoclast differentiation and hyperactivity might represent a promising treatment option. We developed a multifunctional peptide nanocarrier combining bioactive EPI-X4 peptides and the Rho-inhibiting C3bot enzyme from Clostridium botulinum. C3bot is preferentially internalized into the cytosol of monocytic cells, including osteoclasts, where it inhibits Rho-mediated signal transduction. However, Rho-mediated cellular processes like migration and cell division can also be inhibited in non-monocytic cells if C3bot is delivered into their cytosol by a nanocarrier. To accomplish this, we designed a supramolecular transporter where one molecule of biotinylated C3bot and three biotinylated entities of the human EPI-X4 peptide-derived CXCR4 antagonist JM173 are assembled on avidin as a central platform. This modular transport system (JM173)3-Avi-C3 down-modulated osteoclast formation and hyperactivity and delivered the therapeutic cargo C3bot successfully into the cytosol of breast cancer cells, where it inhibited Rho.

Pertussis, also known as whooping cough, is a highly infectious respiratory disease caused by the bacterium Bordetella pertussis. The bacterial virulence factor, pertussis toxin (PT), is associated with the manifestation of the characteristic symptoms of pertussis and the severe form of this disease. Increasing case numbers and the lack of treatment options highlight the need to develop novel pharmacological strategies, e.g., the generation of specific PT inhibitors. Recently, we identified the endogenous human protein α1-antitrypsin (α1AT) as an inhibitor of PT from a screening of a human hemofiltrate protein/peptide library. In the present work, we tested an in-house α1AT peptide bank to identify an α1AT region with anti-PT activity. Then, we compared the sequences of the positive hits from the peptide bank with all known α1AT fragments in the hemofiltrate samples to find new active peptides. In total, 36 peptides were tested for their PT inhibition, leading to the identification of an endogenous α1AT fragment, α1AT HF, derived from hemofiltrate with anti-PT activity. This peptide had no toxic effects on HeLa cells and in vivo on zebrafish embryos, rendering it an attractive lead compound for further evaluation to treat pertussis in the future.

Background: C3-activation products (C3b, iC3b, C3dg) are one major class of opsonins crucial for opsonophagocytosis, which is vital for cellular maintenance and immune surveillance. Complement receptor 3 (CR3; CD11b/CD18, αMβ2) is a key receptor on phagocytes enabling C3-opsonin decorated material to be engulfed efficiently. When engaging certain ligands, CR3 switches from the inactive, bent into an extended, high affinity conformation. Most ligands bind CR3 via its conserved I-domain harboured in the CD11b chain. However, the CyaA toxin from Bordetella pertussis is unique as it binds CR3 outside of its I-domain and prefers the inactive conformation, displaying a non-canonical binding mode1. Interfering with CR3 binding may modulate the inflammatory signature of phagocytes. We hypothesise that CyaA interferes with the recognition of C3-opsonins and consequently alters the activation state of the phagocyte. Methods: Recombinant protein expression was achieved using bacterial, yeast and mammalian cells. Combinations of conservative and tag-based purification strategies were employed for protein purification. Ligand receptor interactions were characterised by surface plasmon resonance (SPR) and spectral shift technology. Results: We expressed the major ligand binding domain αM-I, the complete 6 domain spanning CR3 headpiece and the C-terminal receptor binding part of the CyaA toxin at high purity. Functionality of the purified αM-I domain and the CR3 headpiece was validated by determining binding affinities to C3-opsonins, which are in accordance with published data. The CR3 headpiece showed a 3- to 10-fold higher affinity than αM-I for binding to iC3b: KD Headpiece = 149 nM and KD αM-I = 484 nM for iC3b immobilised on an SPR chip; KD Headpiece = 94.6 nM and KD αM-I = 1.01 μM for interaction with iC3b in the fluid phase. These data support the hypothesis of additional binding sites for iC3b being present outside of the I-domain. The CyaA toxin binding domain bound to the CR3 headpiece (KD = 240 nM) and may proof a valuable tool for receptor competition. Conclusion: The assembly of a recombinantly expressed molecular toolkit for CR3 protein interaction and the successful validation of C3-opsonin and CyaA toxin binding provides a basis for CR3 modulation on the phagocytic cell level. References: Goldsmith, J. A., DiVenere, A. M., Maynard, J. A. & McLellan, J. S. Structural basis for non-canonical integrin engagement by Bordetella adenylate cyclase toxin. Cell Rep. 40, 111196 (2022).

Clostridioides (C.) difficile is a spore-forming, toxin-producing nosocomial human gut pathogen and a causative agent of gastrointestinal infections, leading to mild to severe diarrhea. Severe C. difficile infections (CDI) can cause life-threatening conditions, such as pseudomembranous colitis, colonic perforation, or toxic megacolon. The main virulence factors of C. difficile and responsible for CDI symptoms are two AB-type protein toxins, toxin A (TcdA) and toxin B (TcdB). TcdA and TcdB are large, single-chain proteins with multiple domains and glucosyltransferase activity. After receptor-mediated endocytosis, acidification of endosomes triggers insertion and pore formation of the toxins into the endosomal membrane for the delivery of their toxic glucosyltransferase domain (GTD) into the cytosol. There, the GTD glucosylates its target proteins, small GTPases of the Rho and/or Ras family, which leads amongst others to the collapse of the actin cytoskeleton and eventually to cell death. Here, we describe in silico predicted antimicrobial peptides, denoted as Angies, since they derive from the human endogenous protein angiogenin, as inhibitors for TcdA and TcdB. The strongest inhibitory capacity provided the derivative Angie 5, consistently in HeLa and Vero cells, as well as in the physiologically more relevant colon carcinoma cell line CaCo-2. Angie 5 delayed TcdA/TcdB-mediated glucosylation of its substrate proteins and, consequently, toxin-induced cell rounding as a consequence of actin-depolymerization. Moreover, the same Angie peptides that neutralized TcdA/TcdB also prevented the growth of C. difficile in vitro. In conclusion, our study paves the way for the development of antimicrobial peptide-based anti-toxin strategies to address C. difficile-associated diseases (CDADs).

Shiga toxins (Stx) produced by Shiga toxin-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) are ribosome-inactivating AB5 proteins that consist of one enzymatic active A-subunit (StxA) and a pentamer of non-covalently linked B-subunits (StxB). The description of Stx as an AB5 protein and the observation that A-subunits without their corresponding B-subunits also intoxicate eukaryotic cells, led to the question whether A- and B-subunits are produced in the bacteria in a 1:5 ratio or whether the A-subunit of the clinically most prominent subtype Stx2a is transcribed in excess revealing free A-subunits released in the bacterial environment. The aim of this study was therefore, to investigate the genetic and protein-based background for this observation in six Stx2a-encoding STEC and EHEC wildtype strains. For this purpose, transcriptional analysis of the Stx2a subunit genes, stxA2a and stxB2a, was performed by quantitative real-time PCR in one foodborne O113:H21 STEC isolate (strain TS18/08) and five HUS-associated EHEC strains with the serotypes O157:H7/H- (HUSEC003, HUSEC004), O103:H- (HUSEC008), O26:H11 (HUSEC018), and O104:H4 (LB226692). Contrary to the hypothesis that the A- and B-subunit genes are expressed in a ratio of 1:5 comparable to the holotoxin structure or in a ratio of 1:1 based on the operon structure, the results showed that stxA2a was expressed 1.90 ± 0.55-times stronger than the gene encoding the B-subunit, possibly indicating the presence of free A-subunits. In addition, strain-specific differences regarding the mRNA fold-changes of the A-subunit gene were observed. By use of native polyacrylamide gel electrophoresis and subsequent Western blot analysis, those single A-subunits were indeed detected in the culture supernatants of all six strains. To investigate whether the transcription ratios between A- and B-subunits observed are in a similar range as the amount of subunit proteins present after translation, a quantitative ELISA specific for StxA2a and StxB2a was established. Quantification of the subunits on protein level by use of ELISA revealed that the subunit ratio of StxA2a:StxB2a is 1.10 ± 0.20 for the strains HUSEC003, HUSEC004 and HUSEC008, but 4.63 ± 0.31 for the strains TS18/08, LB226692, and HUSEC018. The results of this study demonstrated that on both, the transcriptional and the translational level, the established 1:5 subunit ratio is not present in all investigated strains. In addition, the ratios observed after translation indicate that in some strains StxA2a subunits are even produced in higher amounts than B-subunits.

Chemoselective dual functionalization of proteins has emerged as an invaluable tool to introduce two distinct payloads to proteins, thus greatly expanding their structural and functional repertoire for more advanced biomedical applications. Here, we introduce N-alkylpyridinium reagents as soft electrophiles for chemoselective dual modification of cysteine residues in peptides or proteins via a 1,6-addition reaction. The N-alkylpyridinium derivatives can be synthesized in two reaction steps revealing good water solubility, high labelling efficiency and chemoselectivity towards cysteine over lysine/N-terminal amine residues, even when used in large excess. This reaction can be combined with strain-promoted azide-alkyne click (SPAAC) and inverse-electron-demand Diels-Alder (iEDDA) reactions to achieve dual functionalization of proteins in a sequential simple one-pot reaction. As a proof-of-concept, the Rho-inhibiting enzyme Clostridium botulinum C3 is functionalized with a cancer cell-targeting peptide and a fluorescent dye for the inhibition of specific Rho-mediated intracellular pathways. The high stability, ease of synthesis, fast reaction kinetics, high water-solubility and chemoselectivity make N-alkylpyridinium reagents unique for dual modification of peptides and proteins to increase their functional diversities for medical applications.

Chronic inflammatory disorders represent one of the predominant healthcare burdens. There is evidence that the oleogum resin from Boswellia serrata trees can downmodulate pro-inflammatory processes. Lipid micellar preparations of Boswellia serrata have been introduced to the market to overcome the low bioavailability of nonformulated Boswellia oleogum resin preparations. In this study, we aimed to compare the anti-inflammatory effects of two different Boswellia serrata nutraceuticals: the native, nonformulated Biotikon® BS-85 and the micellar Boswellia-Loges®. We have previously shown that single oral administration of 800 mg of either formulation reduces the release of proinflammatory cytokines TNF-α, IL-1β, and IL-6 by LPS-activated blood of donors. Here we show that under the same conditions, the production of IL-17A was increased by the nonformulated, native extract of Boswellia serrata oleogum resin. In vitro, the nonformulated but not the micellar formulation of Boswellia serrata oleogum resin decreased the release of IFN-γ, TNF-α, and IL-2 by TCR-activated lymphocytes. Both formulations as well as the bioactive principles boswellic acids lowered NF-κB activity in TCR-activated T lymphocytes. Similarly, both Boswellia serrata formulations and boswellic acids reduced NFAT activity in TCR-activated T lymphocytes. The nonformulated Boswellia serrata extract exhibited higher inhibitory activity on the release of T-cell cytokines. The results suggest that nutraceuticals containing the nonformulated oleogum extract of Boswellia serrata might be more effective in hampering chronic inflammatory disorders characterized by increased activity of T cells than the micellar formulations.

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.

Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.

Many bacteria act pathogenic by the release of AB-type protein toxins that efficiently enter human or animal cells and act as enzymes in their cytosol. This leads to disturbed cell functions and the clinical symptoms characteristic for the individual toxin. Therefore, molecules that directly target and neutralize these toxins provide promising novel therapeutic options. Here, we found that the FDA-approved drug disulfiram (DSF), used for decades to treat alcohol abuse, protects cells from intoxication with diphtheria toxin (DT) from Corynebacterium diphtheria, the causative agent of diphtheria, lethal toxin (LT) from Bacillus anthracis, which contributes to anthrax, and C2 enterotoxin from Clostridium botulinum when applied in concentrations lower than those found in plasma of patients receiving standard DSF treatment for alcoholism (up to 20 µM). Moreover, this inhibitory effect is increased by copper, a known enhancer of DSF activity. LT and C2 are binary toxins, consisting of two non-linked proteins, an enzyme (A) and a separate binding/transport (B) subunit. To act cytotoxic, their proteolytically activated B subunits PA63 and C2IIa, respectively, form barrel-shaped heptamers that bind to their cellular receptors and form complexes with their respective A subunits LF and C2I. The toxin complexes are internalized via receptor-mediated endocytosis and in acidified endosomes, PA63 and C2IIa form pores in endosomal membranes, which facilitate translocation of LF and C2I into the cytosol, where they act cytotoxic. In DT, A and B subunits are located within one protein, but DT also forms pores in endosomes that facilitate translocation of the A subunit. If cell binding, membrane translocation, or substrate modification is inhibited, cells are protected from intoxication. Our results implicate that DSF neither affects cellular binding nor the catalytic activity of the investigated toxins to a relevant extend, but interferes with the toxin pore-mediated translocation of the A subunits of DT, LT and C2 toxin, as demonstrated by membrane-translocation assays and toxin pore conductivity experiments in the presence or absence of DSF. Since toxin translocation across intracellular membranes represents a central step during cellular uptake of many bacterial toxins, DSF might neutralize a broad spectrum of medically relevant toxins.

Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111–132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111–132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure–activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120–132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120–132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111–132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111–132) may offer perspectives to restrict autophagy-dependent viruses.

Silver nanoparticles (AgNPs) conjugated with polymers are well-known for their powerful and effective antimicrobial properties. In particular, the incorporation of AgNPs in biocompatible catecholamine-based polymers, such as polydopamine (PDA), has recently shown promising antimicrobial activity, due to the synergistic effects of the AgNPs, silver(I) ions released and PDA. In this study, we generated AgNPs-PDA-patterned surfaces by localised electrochemical depositions, using a double potentiostatic method via scanning electrochemical cell microscopy (SECCM). This technique enabled the assessment of a wide parameter space in a high-throughput manner. The optimised electrodeposition process resulted in stable and homogeneously distributed AgNP-microspots, and their antimicrobial activity against Escherichia coli was assessed using atomic force microscopy (AFM)-based force spectroscopy, in terms of bacterial adhesion and cell elasticity. We observed that the bacterial outer membrane underwent significant structural changes, when in close proximity to the AgNPs, namely increased hydrophilicity and stiffness loss. The spatially varied antimicrobial effect found experimentally was rationalised by numerical simulations of silver(I) concentration profiles.

Pertussis toxin (PT) is a bacterial AB5-toxin produced by Bordetella pertussis and a major molecular determinant of pertussis, also known as whooping cough, a highly contagious respiratory disease. In this study, we investigate the protective effects of the chaperonin TRiC/CCT inhibitor, HSF1A, against PT-induced cell intoxication. TRiC/CCT is a chaperonin complex that facilitates the correct folding of proteins, preventing misfolding and aggregation, and maintaining cellular protein homeostasis. Previous research has demonstrated the significance of TRiC/CCT in the functionality of the Clostridioides difficile TcdB AB-toxin. Our findings reveal that HSF1A effectively reduces the levels of ADP-ribosylated Gαi, the specific substrate of PT, in PT-treated cells, without interfering with enzyme activity in vitro or the cellular binding of PT. Additionally, our study uncovers a novel interaction between PTS1 and the chaperonin complex subunit CCT5, which correlates with reduced PTS1 signaling in cells upon HSF1A treatment. Importantly, HSF1A mitigates the adverse effects of PT on cAMP signaling in cellular systems. These results provide valuable insights into the mechanisms of PT uptake and suggest a promising starting point for the development of innovative therapeutic strategies to counteract pertussis toxin-mediated pathogenicity.