Production and Characterization of Antibodies against Each of the Three Subunits of the Bacillus cereus Nonhemolytic Enterotoxin Complex

Abstract

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.Bacillus cereus is known to cause two different types of food poisoning (for reviews, see references 9, 15, and 26), which are characterized by either emesis or diarrhea. At present, two different protein complexes, each consisting of three exoproteins, as well as a single protein (cytotoxin K) (19), are discussed as causative agents (9, 13). The enterotoxin described by Beecher and Wong (3, 5), consisting of the components B, L 1 , and L 2 , showed hemolytic activity and was therefore named hemolysin BL (HBL). HBL has been characterized intensively in view of the biological activity (5) as well as genetically (14,25). The nonhemolytic enterotoxin (Nhe) described by Lund and Granum (17) contains the protein components NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa). The genes encoding the components of Nhe have been cloned and characterized, and it has been shown that they are transcribed as one operon (10, 16).Specific monoclonal antibodies (MAbs) for immunochemical studies on the protein level are available only for HBL (6). Due to this limitation, the detection of the B. cereus enterotoxins is still not satisfactory, and a range of in vivo and in vitro tests is used to estimate the toxicity of B. cereus isolates, e.g., the mouse lethality test, the rabbit ileal loop test, the vascular permeability reaction, and cell culture assays (1, 3, 5, 27). These assays, however, do not allow differentiation between the specific activities of the individual toxins. On the other hand, several studies published during recent years showed that nearly all strains of B. cereus harbor nhe, whereas hbl genes were detected in only about 50% of the tested isolates (7,11,12,21,22,28). Although there is some evidence that both c...

Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia

“…In a study of over 100 B. cereus strains, cytotoxicity correlated well with the amount of Nhe produced but poorly with the concentration of Hbl (Moravek et al, 2006). The same group (Dietrich et al, 2005) found that mAb 1E11 raised against NheB prevented cytotoxicity in 20 out of 20 strains of B. cereus isolated from food. In a study of Bacillus thuringiensis strain 407 Cry 2 (indistinguishable from B. cereus), culture supernatants from hbl and cytK deletion mutants showed no impairment in cytotoxicity towards HeLa and Caco-2 cells (Ramarao & Lereclus, 2006), implying that Nhe was the major virulence factor.…”

“…PAGE and Western immunoblotting were carried out as described previously (Lindbäck et al, 2004), using mAbs 1A8 (Dietrich et al, 2005) and 1C2 (Dietrich et al, 1999). Biotin-conjugated goat anti-mouse antibody was used as a secondary antibody (1 : 1000).…”

“…All experiments on cultured cells were carried out in extracellular bathing solution (referred to as EC buffer) containing NaCl (135 mM), HEPES (15 mM), MgCl 2 (1 mM), CaCl 2 (1 mM) and glucose (10 mM), adjusted to pH 7.0-7.1 with Tris. The non-neutralizing mAbs 1A8, raised against NheA (Dietrich et al, 2005), and 1C2, raised against NheB (Dietrich et al, 1999), were used for immunoblotting. mAb 1E11, raised against NheB, is able to neutralize the cytotoxic activity of Nhe, as described by Dietrich et al (2005).…”

“…The non-neutralizing mAbs 1A8, raised against NheA (Dietrich et al, 2005), and 1C2, raised against NheB (Dietrich et al, 1999), were used for immunoblotting. mAb 1E11, raised against NheB, is able to neutralize the cytotoxic activity of Nhe, as described by Dietrich et al (2005).…”

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

“…Vero monkey kidney epithelial cells were grown under standard tissue culture conditions. Basic characteristics of the monoclonal antibody (MAb) 1E11 against NheB and rabbit antiserum (polyclonal antibody [pAb]) against NheC have previously been described (7). At a concentration of 10 g ml Ϫ1 , MAb 1E11 was able to neutralize the cytotoxic activity of the Nhe preparations used in this study.…”