Development and evaluation of cell membrane-based biomimetic nanoparticles loaded by Clostridium perfringens epsilon toxin: a novel vaccine delivery platform for Clostridial-associated diseases.

As Clostridium perfringens (C. perfringens) epsilon toxin (ETX) ranks as the third most potent clostridial toxin after botulinum and tetanus toxins, vaccination is necessary for creatures that can be affected by it to be safe from the effects of this toxin. Nowadays, nanostructures are good choices for carriers for biological environments. We aimed to synthesize biomimetic biodegradable nanodevices to enhance the efficiency of the ETX vaccine. For this purpose, poly(lactic-co-glycolic acid) (PLGA) copolymer loaded with purified epsilon protoxin (proETX) to create nanoparticles called nanotoxins (NTs) and then coated by RBC membrane-derived vesicles (RVs) to form epsilon nanotoxoids (RV-NTs). The resulting RV-NTs shaped smooth spherical surfaces with double-layer core/shell structure with an average particle size of 105.9 ± 35.1 nm and encapsulation efficiency of 97.5% ± 0.13%. Compared with NTs, the RV-NTs were more stable for 15 consecutive days. In addition, although both structures showed a long-term cumulative release, the release rates from RV-NTs were slower than NTs during 144 hours. According to the results of cell viability, ETX loading in PLGA and entrapment in the RBC membrane decreased the toxicity of the toxin. The presence of PLGA enhances the uptake of proETX, and the synthesized structures showed no significant lesion after injection. These results demonstrate that NTs and RV-NTs could serve as an effective vaccine platform to deliver ETX for future in vivo assays.

Direct Visualization of the Dynamic Process of Epsilon Toxin on Hemolysis.

Hemolysis is the process of rupturing erythrocytes (red blood cells) by forming nanopores on their membranes using hemolysins, which then impede membrane permeability. However, the self-assembly process before the state of transmembrane pores and underlying mechanisms of conformational change are not fully understood. In this work, theoretical and experimental evidence of the pre-pore morphology of Clostridium perfringens epsilon toxin (ETX), a typical hemolysin, is provided using in situ atomic force microscopy (AFM) complemented by molecular dynamics (MD) simulations to detect the conformational distribution of different states in Mica. The AFM suggests that the ETX pore is formed in two stages: ETX monomers first attach to the membrane and form a pre-pore in no special conditions required, which then undergo a conformational change to form a transmembrane pore at temperatures above the critical point in the presence of receptors. The authors' MD simulations reveal that initial nucleation occurs when specific amino acids adsorb to negatively charged mica cavities. This work fills the knowledge gap in understanding the early stage of hemolysis and the oligomerization of hemolysins. Moreover, the newly identified pre-pore of ETX holds promise as a candidate for nanopore applications.

Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention.

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.

Structural and biochemical characterization of the Clostridium perfringens-specific Zn2+-dependent amidase endolysin, Psa, catalytic domain.

Phage-derived endolysins, enzymes that degrade peptidoglycans, have the potential to serve as alternative antimicrobial agents. Psa, which was identified as an endolysin encoded in the genome of Clostridium perfringens st13, was shown to specifically lyse C. perfringens. Psa has an N-terminal catalytic domain that is homologous to the Amidase_2 domain (PF01510), and a novel C-terminal cell wall-binding domain. Here, we determined the X-ray structure of the Psa catalytic domain (Psa-CD) at 1.65 Å resolution. Psa-CD has a typical Amidase_2 domain structure, consisting of a spherical structure with a central ß-sheet surrounded by two α-helix groups. Furthermore, there is a Zn2+ at the center of Psa-CD catalytic reaction site, as well as a unique T-shaped substrate-binding groove consisting of two grooves on the molecule surface. We performed modeling study of the enzyme/substrate complex along with a mutational analysis, and demonstrated that the structure of the substrate-binding groove is closely related to the amidase activity. Furthermore, we proposed a Zn2+-mediated catalytic reaction mechanism for the Amidase_2 family, in which tyrosine constitutes part of the catalytic reaction site.

A Novel PilR/PilS Two-Component System Regulates Necrotic Enteritis Pilus Production in Clostridium perfringens.

Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE pilus), along with a two-component system (TCS) designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS in the regulation of pilin production. Isogenic pilR, agrB, and virR null mutants were generated from the parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1pilR mutant, while production in its complement (CP1pilR+) was greater than wild-type levels. In contrast, pilus production in the agrB and virR mutants was comparable or higher than the wild type but reduced in their respective complemented strains. When examined for collagen-binding activity, the pilR mutant showed significantly lower binding to most collagen types (types I to V) than parental CP1 (P ≤ 0.05), whereas this activity was restored in the complemented strain (P > 0.05). In contrast, binding of agrB and virR mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 (P > 0.05), whereas the complemented strains exhibited significantly reduced binding (P ≤ 0.05). These data suggest the PilRS TCS positively regulates pilus production in C. perfringens, while the Agr-like QS system may serve as a negative regulator of this operon. IMPORTANCE Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the postantibiotic era. Multiple factors in C. perfringens, including both virulent and nonvirulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development, along with a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens. In addition, the Agr-like quorum sensing signaling pathway was found to be involved in the regulation. These findings have identified additional targets for developing nonantibiotic strategies to control NE disease.

O-GlcNAcylation Quantification of Certain Protein by the Proximity Ligation Assay and Clostridium perfringen OGAD298N(CpOGAD298N).

O-GlcNAcylation is an O-linked ß-N-acetyl-glucosamine (O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays a vital role in many critical cellular processes. Owing to its low molecular weight, uncharged property, and difficulty in distinguishing from ß-N-acetyl-galactosamine (GalNAc), the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions. Here, we compared glycan array data of the mutant of Clostridium perfringen OGA (CpOGAD298N), O-GlcNAc antibody CTD110.6, and several lectins. We found that CpOGAD298N can effectively distinguish GlcNAc from GalNAc. Glycan array analysis and isothermal titration calorimetry (ITC) show that CpOGAD298N has a GlcNAc specific binding characteristic. CpOGAD298N could be used in far-western, flow cytometry analysis, and confocal imaging to demonstrate the existence of O-GlcNAc proteins. Using the CpOGAD298N affinity column, we identified 84 highly confident O-GlcNAc modified peptides from 82 proteins in the MCF-7 cell line and 33 highly confident peptides in 33 proteins from mouse liver tissue; most of them are novel O-GlcNAc proteins and could not bind with wheat germ agglutinin (WGA). Besides being used as a facile enrichment tool, a combination of CpOGAD298N with the proximity ligation assay (PLA) is successfully used to quantify O-GlcNAc modified histone H2B, which is as low as femtomoles in MCF-7 cell lysate. These results suggest that CpOGAD298N is a specific tool for detection (far-western, flow cytometry analysis, and confocal imaging) and enrichment of O-GlcNAcylated proteins and peptides, and the CpOGAD298N-PLA method is useful for quantifying certain O-GlcNAc protein.

Bioinformatics analysis of NetF proteins for designing a multi-epitope vaccine against Clostridium perfringens infection.

Clostridium perfringens is an important human and animal pathogen that is the primary causative agent of necrotizing enteritis and enterotoxemia in many types of animals. C. perfringens produces a variety of toxins, including NetF which may plays a crucial role in the pathogenesis of foal and canine necrotizing enteritis. In this study, we used several bioinformatics methods to analyze various aspects of the NetF proteins, including the physicochemical properties, secondary and tertiary structures, and the dominant B-cell and T-cell epitopes. The results showed that NetF protein was a stable and hydrophilic protein. The secondary structure of the NetF protein consisted of 2.62% alpha helixes, 6.56% beta turns, 38.69% extended strands and 52.13% random coils. Moreover, several potential B and T-cell epitopes were identified for NetF. In addition, the obtained findings from antigenicity and allergenicity evaluation remarked that this protein is immunogenic and non-allergen. Based on the results of Ramachandran plot, 94.22%, 5. 42%, and 0.36% of amino acid residues were incorporated in the favored, allowed, and outlier regions, respectively. This study provides a foundation for further investigations, and laid a theoretical basis for the development of an appropriate vaccine against C. perfringens infection.

An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response.

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(ß1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

Central residues of the amphipathic ß-hairpin loop control the properties of Clostridium perfringens epsilon-toxin channel.

Clostridium perfringens epsilon toxin (ETX) is a heptameric pore-forming toxin of the aerolysin toxin family. ETX is the most potent toxin of this toxin family and the third most potent bacterial toxin with high cytotoxic and lethal activities in animals. In addition, ETX shows a demyelinating activity in nervous tissue leading to devastating multifocal central nervous system white matter disease in ruminant animals. Pore formation in target cell membrane is most likely the initial critical step in ETX biological activity. Eight single to quadruple ETX mutants were generated by replacement of polar residues (serine, threonine, glutamine) in middle positions of the ß-strands forming the ß-barrel and facing the channel lumen with charged glutamic residues. Channel activity and ion selectivity were monitored in artificial lipid monolayer membranes and cytotoxicity was investigated in MDCK cells by the viability MTT test and propidium iodide entry. All the mutants formed channels with similar conductance in artificial lipid membranes and increasing cation selectivity for increasing number of mutations. Here, we show that residues in the central position of each ß-strand of the amphipathic ß-hairpin loop that forms the transmembrane pore, control the size and ion selectivity of the channel. While the highest cationic ETX mutants were not cytotoxic, no strict correlation was observed between ion selectivity and cytotoxicity.

Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex.

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.

Near-infrared spectroscopy coupled with chemometrics algorithms for the quantitative determination of the germinability of Clostridium perfringens in four different matrices.

Clostridium perfringens (C. perfringens) has the ability to form metabolically-dormant spores that can survive food preservation processes and cause food spoilage and foodborne safety risks upon germination outgrowth. This study was conducted to investigate the effects of different AGFK concentrations (0, 50, 100, 200 mM/mL) on the spore germination of C. perfringens in four matrices, including Tris-HCl, FTG, milk, and chicken soup. C. perfringens spore germinability was investigated using near infrared spectroscopy (NIRS) combined with chemometrics. The spore germination rate (S), the OD600%, and the Ca2+-DPA% were measured using traditional spore germination methods. The results of spore germination assays showed that the optimum germination rate was obtained using 100 mM/L concentrations of AGFK in the FTG medium, and the S, OD600% and Ca2+-DPA% were 98.6%, 59.3% and 95%, respectively. The best prediction models for the S, OD600% and Ca2+-DPA% were obtained using SNV as the preprocessing method for the original spectra, with the competitive adaptive weighted resampling method (CARS) as the characteristic variables related to the selected spore germination methods from NIRS data. The results of the S showed that the optimum model was built by CARS-PLSR (RMSEV = 0.745, Rc = 0.897, RMSEP = 0.769, Rp = 0.883). For the OD600%, interval partial least squares regression (CARS-siPLS) was performed to optimize the models. The calibration yielded acceptable results (RMSEV = 0.218, Rc = 0.879, RMSEP = 0.257, Rp = 0.845). For the Ca2+-DPA%, the optimum model with CARS-siPLS yielded acceptable results (RMSEV = 44.7, Rc = 0.883, RMSEP = 50.2, Rp = 0.872). This indicated that quantitative determinations of the germinability of C. perfringens spores using NIR technology is feasible. A new method based on NIR was provided for rapid, automatic, and non-destructive determination of the germinability of C. perfringens spores.

A Method to Prepare Claudin-Modulating Recombinant Proteins.

The epithelium forms tight junctions by sealing the paracellular space, and tight junctions prevent the free movement of solutes. Claudin is an important structural and functional component of tight junctions and contributes to the formation of paracellular pathways for different populations of size- and charge-selective solutes. Therefore, modulation of tight junctions is important to develop drug delivery strategies. Clostridium perfringens enterotoxin (CPE) causes food poisoning in humans and is a 35-kDa polypeptide, consisting of 319 amino acids and two functional regions. The C-terminal region of CPE (C-CPE) is not cytotoxic and binds to its receptor claudin, which in turn modulates the epithelial tight junction barrier. Thus, claudin binders, such as C-CPE, are useful tools for drug delivery targeting tight junctions. Here, we provide a protocol for the expression and purification of recombinant C-CPE proteins as claudin binders, an analysis method for C-CPE binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.

Structures of major pilins in Clostridium perfringens demonstrate dynamic conformational change.

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.

Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus.

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

Study of the Structure and Biological Activity of the Amino-Terminus of the α-Toxin from Clostridium welchii Type A.

To explore the biological activity of Clostridium welchii α-toxin (CPA), the Asp56 residue of CPA was mutated to glycine (CPA D56G) by site-directed mutagenesis, and the 250 amino acid amino-terminal phospholipase C (PLC)-containing domain of CPA (PLC1-250) was isolated. The secondary and three-dimensional (3D) structures of CPA D56G and PLC1-250 were predicted, and the results showed that the secondary structures of CPA D56G and PLC1-250 were composed of α-helices and random coils. The 3D structures of CPA D56G and PLC1-250 were similar to the 3D structures of CPA. The circular dichroism (CD) spectrum of CPA D56G differed from the CD spectrum of CPA, but the CD spectrum of PLC1-250 was similar to the CD spectrum of CPA. Biological activity assays showed that CPA D56G lost the PLC activity of CPA and that mice immunized with CPA D56G were protected against a challenge with 1 MLD C. welchii type A strain C57-1. In addition, PLC1-250 contained the PLC activity of CPA. This study laid a solid foundation for future studies on the relationship between the molecular structure and biological function of CPA and its molecular mechanism. Our study also provided CPA D56G as a candidate strain for engineering a CPA subunit vaccine for C. welchii type A.

Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown.

Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJ-forming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13× lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.

Fine-tuning of the stability of ß-strands by Y181 in perfringolysin O directs the prepore to pore transition.

Perfringolysin O (PFO) is a toxic protein that forms ß-barrel transmembrane pores upon binding to cholesterol-containing membranes. The formation of lytic pores requires conformational changes in PFO that lead to the conversion of water-soluble monomers into membrane-bound oligomers. Although the general outline of stepwise pore formation has been established, the underlying mechanistic details await clarification. To extend our understanding of the molecular mechanisms that control the pore formation, we compared the hydrogen-deuterium exchange patterns of PFO with its derivatives bearing mutations in the D3 domain. In the case of two of these mutations F318A, Y181A, known from previous work to lead to a decreased lytic activity, global destabilization of all protein domains was observed in their water-soluble forms. This was accompanied by local changes in D3 ß-sheet, including unexpected stabilization of functionally important ß1 strand in Y181A. In case of the double mutation (F318A/Y181A) that completely abolished the lytic activity, several local changes were retained, but the global destabilization effects of single mutations were reverted and hydrogen-deuterium exchange (HDX) pattern returned to PFO level. Strong structural perturbations were not observed in case of remaining variants in which other residues of the hydrophobic core of D3 domain were substituted by alanine. Our results indicate the existence in PFO of a well-tuned H-bonding network that maintains the stability of the D3 ß-strands at appropriate level at each transformation step. F318 and Y181 moieties participate in this network and their role extends beyond their direct intermolecular interaction during oligomerization that was identified previously.

Calcium enhances binding of Clostridium perfringens epsilon toxin to sulfatide.

Epsilon toxin (Etx) from Clostridium perfringens is synthesized as a very low-active prototoxin form (proEtx) that becomes active upon proteolytic activation and has the capacity to cross the blood-brain barrier (BBB), thereby producing severe neurological effects. The identity and requirements of host receptors of Etx remain a matter of controversy. In the present study, we analysed the binding of proEtx or Etx to liposomes containing distearoylphosphatidylcholine (DSPC), cholesterol and sulfatide, or alternatively to detergent-solubilized lipids, using surface plasmon resonance (SPR). We also tested the influence of calcium on Etx or proEtx binding. Our findings show that the presence of sulfatide in liposomes increases both Etx and proEtx binding, and Etx binding is enhanced by calcium. These results were corroborated when SPR was conducted with immobilized toxin, since detergent-solubilized sulfatide increases its binding to Etx in the presence of calcium, but not to proEtx. Moreover, binding affinity is also affected, since the treatment of liposomes with sulfatase causes the dissociation rate constants (KD) in both proEtx and Etx to increase, especially in the case of proEtx in the presence of calcium. In addition, protein-lipid overlay assays corroborated the calcium-induced enhancement of Etx binding to sulfatide, and to lipids extracted from sulfatide-enriched rat brain lipid rafts. In conclusion, the present work highlights the role of sulfatide as an important element in the pathophysiology of Etx and reveals the influence of calcium in the interaction of Etx, but not of proEtx, with the target membrane.

Functionalization of gold-nanoparticles by the Clostridium perfringens enterotoxin C-terminus for tumor cell ablation using the gold nanoparticle-mediated laser perforation technique.

A recombinant produced C-terminus of the C. perfringens enterotoxin (C-CPE) was conjugated to gold nanoparticles (AuNPs) to produce a C-CPE-AuNP complex (C-CPE-AuNP). By binding to claudins, the C- CPE should allow to target the AuNPs onto the claudin expressing tumor cells for a subsequent cell killing by application of the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. Using qPCR and immunocytochemistry, we identified the human Caco-2, MCF-7 and OE-33 as well as the canine TiHoDMglCarc1305 as tumor cells expressing claudin-3, -4 and -7. Transepithelial electrical resistance (TEER) measurements of Caco-2 cell monolayer showed that the recombinant C-CPE bound to the claudins. GNOME-LP at a laser fluence of 60 mJ/cm2 and a scanning speed of 0.5 cm/s specifically eliminated more than 75% of claudin expressing human and canine cells treated with C-CPE-AuNP. The same laser fluence did not affect the cells when non-functionalized AuNPs were used. Furthermore, most of the claudin non-expressing cells treated with C-CPE-AuNP were not killed by GNOME-LP. Additionally, application of C-CPE-AuNP to spheroids formed by MCF-7 and OE-33 cells grown in Matrigel reduced spheroid area. The results demonstrate that specific ablation of claudin expressing tumor cells is efficiently increased by activated C-CPE functionalized AuNPs using optical methods.