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The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

Didier A, Jeßberger N, Krey V, Dietrich R, Scherer S, Märtlbauer E - Toxins (Basel) (2015)

Bottom Line: The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection.Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody.Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians Universität München, 85763 Oberschleißheim, Germany. a.didier@mh.vetmed.uni-muenchen.de.

ABSTRACT
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

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(A) Partial NheB sequences comprising amino acid 121–180. The complete Clustal Omega alignment file is given as Figure S1. Although several primers were applied, sequencing of MHI 3225 was only partially possible for unknown reasons. The most prominent difference is the point mutation at position E151D compared to the reference strain MHI 241. This mutation is not present in a formerly-published rNheB fragment, which always tested negative in EIA and Western blot; (B) position of the aspartic acid residue in the structural model of NheB; and (C) the glutamic acid residue is protuding more and slightly rotated in the model.
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toxins-07-04655-f001: (A) Partial NheB sequences comprising amino acid 121–180. The complete Clustal Omega alignment file is given as Figure S1. Although several primers were applied, sequencing of MHI 3225 was only partially possible for unknown reasons. The most prominent difference is the point mutation at position E151D compared to the reference strain MHI 241. This mutation is not present in a formerly-published rNheB fragment, which always tested negative in EIA and Western blot; (B) position of the aspartic acid residue in the structural model of NheB; and (C) the glutamic acid residue is protuding more and slightly rotated in the model.

Mentions: Results were subsequently translated to the amino acid sequence and compared by the Clustal Omega program [18] (Figure 1A). The most prominent difference consistently found in all six strains showing a uncommonly weak 2B11 reactivity was a single amino acid (aa) exchange at position 151 (E151D). In Figure 1B,C this position is highlighted in the structural model of NheB. The recently-resolved structure of NheA (PDB-ID: 4K1P) [19] served as a template to create a NheB model on the SWISS-MODEL server (Swiss Institute of Bioinformatics and University of Basel, Basel, Switzerland).


The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

Didier A, Jeßberger N, Krey V, Dietrich R, Scherer S, Märtlbauer E - Toxins (Basel) (2015)

(A) Partial NheB sequences comprising amino acid 121–180. The complete Clustal Omega alignment file is given as Figure S1. Although several primers were applied, sequencing of MHI 3225 was only partially possible for unknown reasons. The most prominent difference is the point mutation at position E151D compared to the reference strain MHI 241. This mutation is not present in a formerly-published rNheB fragment, which always tested negative in EIA and Western blot; (B) position of the aspartic acid residue in the structural model of NheB; and (C) the glutamic acid residue is protuding more and slightly rotated in the model.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663526&req=5

toxins-07-04655-f001: (A) Partial NheB sequences comprising amino acid 121–180. The complete Clustal Omega alignment file is given as Figure S1. Although several primers were applied, sequencing of MHI 3225 was only partially possible for unknown reasons. The most prominent difference is the point mutation at position E151D compared to the reference strain MHI 241. This mutation is not present in a formerly-published rNheB fragment, which always tested negative in EIA and Western blot; (B) position of the aspartic acid residue in the structural model of NheB; and (C) the glutamic acid residue is protuding more and slightly rotated in the model.
Mentions: Results were subsequently translated to the amino acid sequence and compared by the Clustal Omega program [18] (Figure 1A). The most prominent difference consistently found in all six strains showing a uncommonly weak 2B11 reactivity was a single amino acid (aa) exchange at position 151 (E151D). In Figure 1B,C this position is highlighted in the structural model of NheB. The recently-resolved structure of NheA (PDB-ID: 4K1P) [19] served as a template to create a NheB model on the SWISS-MODEL server (Swiss Institute of Bioinformatics and University of Basel, Basel, Switzerland).

Bottom Line: The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection.Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody.Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians Universität München, 85763 Oberschleißheim, Germany. a.didier@mh.vetmed.uni-muenchen.de.

ABSTRACT
The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

Show MeSH
Related in: MedlinePlus