Immunological characterization of the subunits of type A botulinum neurotoxin and different components of its associated proteins

Abstract

Botulinum neurotoxins (BoNTs) constitute a family of seven structurally similar but antigenically distinct proteins produced by different strains of Clostridium botulinum. Type A botulinum neurotoxin (BoNT/A) is produced along with 6 neurotoxin associated proteins (NAPs) including hemagglutinin (Hn-33) through polycistronic expression of a clustered group of genes to form a complex (BoNT/AC). The presence of NAPs enhances the oral toxicity of the neurotoxin significantly. Hn-33 makes up the largest fraction of NAPs in BoNT/AC and strongly protects BoNT/A against proteases of the GI tract. BoNT in its complex form is also used in therapeutic and cosmetic applications to treat several neuromuscular disorders. In this study immunological reactivity of BoNT/A in its purified and complex forms, neurotoxin associated proteins, and Hn-33 have been examined using enzyme-linked immunosorbent assay (ELISA). Antibodies raised against the whole complex reacted 60 times better with the complex and 35 times better with Hn-33 and NAPs compared to the purified neurotoxin suggesting stronger immunogenicity of NAPs over that of purified neurotoxin and a higher potential of BoNT/AC and its associated proteins to induce host immune response. This observation also suggests that Hn-33 and other NAPs could potentially be employed as adjuvants for development of vaccines against botulism and could be a good surrogate for botulinum diagnostics. ELISA binding curves of BoNT/AC and BoNT/A with antibodies raised against BoNT/A indicate that BoNT/A in its purified and complex forms induces equal immunogenic response and a 2.5-fold higher immunogenic response compared to BoNT/A light and heavy chains. We have also discovered a new protein, an intimin analog, present within the complex preparation of BoNT/A which shows dramatically high immunoreactivity.

Introduction

Botulinum neurotoxins (BoNTs) are a group of large proteins produced by Clostridium botulinum in seven immunologically distinct serotypes named A–G. BoNTs being the causative agents of the most dreaded food poisoning disease, botulism, are the most potent toxins known to human kind (Singh, 2000). Interestingly, however, BoNT also remarkably serves as a powerful tool to treat a myriad of neuromuscular disorders, and in cosmetic applications (Klien, 2004, Bhidayasiri and Truong, 2005).

BoNTs are produced as ∼150 kDa nontoxic single chain proteins that are activated by proteolytic cleavage to a dichain structure consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) linked through a disulfide bond. To produce its toxic effect, the toxin progresses through a series of well defined steps that includes binding to receptors on the surface of cholinergic nerve endings mediated by the C-terminus of heavy chain, internalization via receptor mediated endocytosis, penetrating the endosome by pH induced translocation, and finally acting enzymatically in the cytosol to block neurotransmitter release. BoNTs possess Zn²⁺ endopeptidase activity against a select group of neuronal proteins involved in the exocytosis process, causing the blockage of acetylcholine release at the neuromuscular junction (Montecucco and Schiavo, 1995, Dressler and Saberi, 2005).

The most common source of botulism is by ingestion of food contaminated with spores of C. botulinum, preserved under anaerobic conditions that favor germination of spores, and secretion of the neurotoxin (Chen et al., 1998). BoNTs are secreted from the bacterium along with a group of neurotoxin associated proteins (NAPs) in the form of a complex. The size of the complex varies with serotypes, but all the serotypes of botulinum neurotoxin complex contain 1–7 additional associated proteins (Sakaguchi, 1983, Cai et al., 1999). A close functional relationship between BoNT and NAPs is strongly indicated by clustering of genes for BoNT, NAPs and a regulatory gene botR (Cai et al., 1999). NAPs have been known to contribute significantly to the oral toxicity of BoNTs (Li et al., 1998). NAPs also have been recently shown to play a critical role in enhancing the endopeptidase activity of the neurotoxin (Sharma and Singh, 2004, Kukreja and Singh, 2007). Because of the extreme toxicity and stability of BoNT in the presence of NAPs, BoNT complexes are on the top of the list of biological warfare agents (Sharma and Singh, 2004). Ironically, it is the complex form of the BoNT along with NAPs that is most commonly used as therapeutic agent to treat several neuromuscular disorders and in cosmetic applications (Shukla and Sharma, 2005).

Specific interactions between the neurotoxin and NAPs in botulinum complex seem to protect the neurotoxin from the acidity and proteases in the GI tract and in the absorption of the toxin in the gut wall (Fu et al., 1998, Fujinaga et al., 1997). At the same time, such protection could interfere with the interaction of an antibody with the neurotoxin when present in the complex form. Therefore, it is important not only to analyze the interference of the NAPs in the detection of the neurotoxin, but also to characterize the immunological properties of the associated proteins themselves. So far, studies have been conducted only on the effect of BoNT at the nerve cell in blocking the release of neurotransmitters to cause flaccid paralysis. BoNT and NAPs could have an effect on other vital systems, in addition to paralysis. Understanding the effect of BoNT and NAPs on the immune system is of particular concern. Thus immunological characterization of BoNT and its associated proteins is critical for not only designing detection systems for this class A bioterror agent, but also for developing more efficient approaches for vaccine development against botulism, and to better understand their immune response in the host.

In this study we have investigated the immunochemical reactivities of BoNT/A in its pure and complex forms, and its associated proteins, to polyclonal antibodies raised against the toxoids of the neurotoxin complex, pure neurotoxin, and its constituent light and heavy chains, and Hn-33 using enzyme-linked immunosorbent assay.

Results indicate that the neurotoxin is accessible to antibodies even in the complex form, but such accessibility is hindered by the presence of NAPs. In addition, antibodies raised against the whole complex have higher reactivity to the complex and NAPs when compared to the purified neurotoxin, suggesting stronger immunogenicity of the NAPs over that of the purified neurotoxin. Higher immunogenicity of the neurotoxin complex is primarily due to the presence of Hn-33. BoNT/AC, NAPs and Hn-33 have a significantly higher potential to induce host response.