Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Abstract

Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected. Botulinum neurotoxins (BoNTs) are produced by anaerobic, Gram-positive, spore-forming bacteria, including Clostridium botulinum and rare strains of Clostridium baratii and Clostridium butyricum. BoNTs are the most poisonous protein toxins known in nature, and they are classified as tier 1 category A select agents due to their potential use as bioterrorism agents (1). Purified BoNT is a protein with a molecular mass of ca. 150 kDa, consisting in its active form of a heavy chain (Hc) (ϳ100 kDa) and a light chain (Lc) (ϳ50 kDa) linked by a disulfide bond and noncovalent molecular interactions (2). The heavy chain contains two functional domains, a receptor binding domain and a translocation domain (3, 4), while the light chain is responsible for catalytic activity on SNARE substrates in the neuronal cell cytosol. BoNTs are distinguished immunologically into seven serotypes designated A to G based on toxin neutralization using homologous antitoxins (5). Recently, the existence of an eighth serotype, H, in a dual-toxin-producing strain of C. botulinum has been reported (5).Of the seven established serotypes, BoNT/A is of particular interest since it is the most potent BoNT, exhibits longer duration in humans than other serotypes, and is also implicated in most cases of human botulism in the continental United States (6, 7). Within serotype A, five subtypes of BoNT/A (A1 to A5) h...

Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX‐type gene cluster

“…In several studies, no stable complex of BoNT/ A2 with any OrfX-protein has been found. Only the M-PTC consisting of BoNT and NTNH has been identified [2,26,52]. This was also the case in this study using recombinant proteins (Fig.…”

A neurotoxin that specifically targets Anopheles mosquitoes

“…2A). High molecular complexes from Pbm were concentrated 21 and the sample, which contained PMP1 and Cry16A (Supplementary Fig. 2B), was separated by native PAGE, subjected to analysis by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS), and compared with a similar extracted fraction from the Pbm∆109 mutant (Supplementary Fig.…”

“…Pb malaysia and Pbm∆109 proteins present in the culture supernatant were acid precipitated adding H 2 SO 4 dropwise to pH 3.5 as described 21 . Precipitated proteins were extracted by agitation for 2 h in 0.1 M sodium citrate buffer pH 5.5 and analyzed in native protein acrylamide gels.…”

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

“…In strain IBCA10-7060/CDC69016, the bont /B2 gene is arranged in an ntnh-ha toxin gene cluster encoding nontoxic nonhemagglutinin and three hemagglutinins (HAs), whereas the BoNT/FA gene is arranged in an ntnh-orfX cluster encoding NTNH, P47, and putatively three ORFX proteins ( 23 ). It has previously been shown that immunoprecipitation of BoNTs with OrfX gene clusters detected only the full-length NTNH in association with the BoNT but not the OrfX proteins, whereas the BoNTs from HA gene clusters contained the NTNH and HA proteins ( 45 ). Thus, the association of BoNT/FA with only NTNH and the elution of the HA proteins in a separate fraction ( Fig.…”

“…For immunoprecipitation analysis, culture supernatants of strain CDC69016/B2tox − were analyzed as described previously ( 45 ). Briefly , polyclonal antibodies raised against BoNT/A1 or BoNT/B1 ( 49 ) were used for immunoprecipitation of the respective toxins from 1 ml of culture supernatants.…”