Cytotoxicity of the
            <i>Bacillus cereus</i>
            Nhe Enterotoxin Requires Specific Binding Order of Its Three Exoprotein Components

Lindbäck, Toril; Hardy, Simon P.; Dietrich, Richard; Södring, Marianne; Didier, Andrea; Moravek, Maximilian; Fagerlund, Annette; Böck, Stefanie; Nielsen, Carina; Casteel, Maximilian; Granum, Per Einar; Märtlbauer, Erwin

doi:10.1128/iai.00247-10

Cited by 64 publications

(93 citation statements)

References 27 publications

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“…NheB and NheC are mostly ␣-helical molecules with a predicted ␤-tongue, showing structural similarities to ClyA (11). The region of the predicted ␤-tongue in NheC is necessary for cell binding but not for interaction with NheB in solution (21). Binding between NheB and NheC could be demonstrated in solution, and both components are able to bind to cell membranes.…”

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confidence: 88%

“…It is a three-component toxin and consists of the exoproteins NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa) (14). Studies using cell-based tests to assay Nhe-specific cytotoxicity demonstrated toxic effects in Vero, GH4, and CaCo-2 cells (17,20,21). The susceptibility of other cell lines has not yet been tested.…”

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confidence: 99%

“…In contrast, NheA does not bind to cells per se, nor does it seem to interact with NheB and NheC in solution. The presence of NheA is, however, mandatory in the final step of the Nhe mode of action in order to trigger toxicity, indicating a specific binding order of the individual components (21).…”

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Monoclonal Antibodies Neutralize Bacillus cereus Nhe Enterotoxin by Inhibiting Ordered Binding of Its Three Exoprotein Components

et al. 2012

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The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB. Bacillus cereus is a major food-borne pathogen known to produce a range of cytotoxins (for reviews, see references 27 and 28). There are four major toxins involved in food poisoning cases, namely, the emetic toxin (cereulide), a dodecadepsipeptide (1), and the three-component diarrheal toxins hemolysin BL (Hbl) (4, 5) and nonhemolytic enterotoxin (Nhe) (23). In addition, a single-component protein toxin (cytotoxin K) causing severe necrotic enteritis was identified in a rare B. cereus strain (22), for which the name "Bacillus cytotoxis" has been proposed (19). Studies on the prevalence of the nhe and hbl genes (10,15,16,26,31) in B. cereus indicate that all strains of B. cereus possess the genes of at least one of the diarrheal enterotoxins, and Nhe is the most prevalent enterotoxin harbored by B. cereus. In addition, the overall B. cereus-associated cytotoxic activity is correlated with the Nhe expression level (24).Nhe was first identified in strain NVH 0075/95, which was isolated following a large food-poisoning outbreak in Norway (23). It is a three-component toxin and consists of the exoproteins NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa) (14). Studies using cell-based tests to assay Nhe-specific cytotoxicity demonstrated toxic effects in Vero, GH4, and CaCo-2 cells (17,20,21). The susceptibility of other cell lines has not yet been tested. It is known that Nhe has intrinsic pore-forming capacity (11) and that maximum toxicity will be reached when the ratio of the individual components is 10:10:1 for NheA, NheB, and NheC, respectively (20). NheB and NheC are mostly ␣-helical molecules with a predicted ␤-tongue, showing structural similarities to ClyA (11). The region of the predicte...

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Monoclonal Antibodies Neutralize Bacillus cereus Nhe Enterotoxin by Inhibiting Ordered Binding of Its Three Exoprotein Components

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“…In particular the mode of action of Nhe on the cell membranes has been described (Lindbäck et al, 2010). Genes coding for Nhe, unlike those coding for Hbl, are present in most, if not all, strains of B. cereus (Guinebretière et al, 2010, Fagerlund et al, 2007 and the amount of Nhe produced at 32 °C by B. cereus strains was correlated with their cytotoxic activities (Moravek et al, 2006).…”

Section: Assessment Of Species Belonging To the Bacillus Cereus Groupmentioning

confidence: 99%

Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition

Feed¹

2014

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Bacillus species are used in animal production directly as microbial feed additives or as the source of other feed additives, notably enzymes. The principal safety concern for consumers and, to a lesser extent livestock, associated with Bacillus is a capacity for toxin production. However, the capacity for toxin production and the nature of the toxins produced is unevenly distributed over the genus, occurring frequently in some species and more rarely in others. In principle, the selection of strains belonging to the B. cereus taxonomic group for direct use in animal production is considered inadvisable. If, however, they are proposed then the full genome should be sequenced and a bioinformatic analysis made to search for genes coding for enterotoxins and cereulide synthase. If there is evidence of homology, the non-functionality of the genes (e.g. mutation, deletion) must be demonstrated. For other species, concerns appear to be associated to the production of surfactin likelipopeptides, although the relation between the presence of these compounds and/or other toxic factors and the risk of illness in human has not yet been established. In the absence of animal models shown to be able to distinguish hazardous from non hazardous strains, the FEEDAP Panel relies on the use of in vitro cell-based methods to detect evidence of a cytotoxic effect. Such tests should be made with culture supernatants since the concentration of cells obtained in a broth culture would always exceed that found in animal food products. If the strain proves to be cytotoxic it is not recommended for use.

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“…During this step NheB undergoes a conformational change allowing association of NheA (step 3), which finally leads to cell lysis. 12 This self-assembly of the three Nhe proteins, triggered by the contact with the cell membrane, forms a natural model of sequential logic and allows the set-up of an intelligent keypad lock ( Fig. 1d and e).…”

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Ordered self-assembly of proteins for computation in mammalian cells

et al. 2014

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A cellular logic system capable of combinatorial and sequential logic operations based on bacterial protein-triggered cytotoxicity was constructed. Advanced devices such as a keypad lock, half-adder and several basic Boolean properties were demonstrated on the cells.Recent advances in our understanding of biology are critical to the development of future biocomputers, with several distinct advantages, such as flexible design, efficient energy usage, memory functions and error checking.1,2 These developed biocomputing systems can be generally grouped into two categories, the bottomup approach uses biomolecules for sensing and performing computation; on the other hand, the top-down method utilizes whole cells including prokaryotic or mammalian cells to implement sophisticated tasks. [3][4][5] For example, bacterial virulence proteins have been used as valuable synthetic biology tools to engineer mammalian cells for therapeutic and analytical applications. 6Compared to the silicon-based computers using sole electrical signals for information processing, natural cellular biocomputing systems harness various inputs including chemicals, biomolecules and even physical factors such as light and heat. After the extracellular inputs have been sensed by cells, they pass through the first barrier, the cell membrane, then activate downstream effectors in the cytoplasm, and eventually trigger different gene regulators in the nucleus leading to the expression of the corresponding proteins, which will execute specific tasks.7 All these events take part in the integral information flow from Input to Output, as shown in Scheme 1a. Whereas the information flow from DNA to RNA and to protein synthesis, according to the central dogma of molecular biology, is well known, 8 the crucial role of the cell membrane, the environment-cell interface, in sensing, filtering, amplification, and storage of external signals is still not fully understood. The environment-cell interface shows a phospholipid bilayer architecture to which various receptors and other recognition elements interacting with extracellular stimuli are anchored. 9 Elucidation of the process of information transduction between Input and the environmentcell interface will facilitate the development of biocomputers with useful human-machine interfaces. In this work, we utilize the selfassembly of a bacterial toxin complex on the cell membrane of immortalized mammalian cells as a 3-input framework to conduct different logic operations (Scheme 1b). This extracellular protein complex, the nonhemolytic enterotoxin (Nhe), is expressed by most strains of Bacillus cereus, but can also be produced by non-B. cereus strains. 10 The Nhe complex consists of the cytolytic protein NheA and the binding components NheB and NheC. Nhe has some unique properties: (i) the individual Nhe proteins are not toxic, (ii) toxicity can be evoked only by a specific ordered binding sequence, provided that (iii) the Nhe proteins are present in a specific molar ratio, and (iv) Nhe toxicity can be neutralize...

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Cytotoxicity of the Bacillus cereus Nhe Enterotoxin Requires Specific Binding Order of Its Three Exoprotein Components

Abstract: This study focuses on the interaction of the three components of the Bacillus cereus

Cited by 64 publications

References 27 publications

Monoclonal Antibodies Neutralize Bacillus cereus Nhe Enterotoxin by Inhibiting Ordered Binding of Its Three Exoprotein Components

Monoclonal Antibodies Neutralize Bacillus cereus Nhe Enterotoxin by Inhibiting Ordered Binding of Its Three Exoprotein Components

Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition

Ordered self-assembly of proteins for computation in mammalian cells

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