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Appl Environ Microbiol. 2010 January; 76(1): 40–47.
Published online 2009 November 13. doi:  10.1128/AEM.01882-09
PMCID: PMC2798633

Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex [down-pointing small open triangle]


Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most potent toxins known in nature and are characterized as category A select agents since they are considered potential bioterrorism threats (3). BoNTs can be distinguished immunologically into seven serotypes by using homologous antitoxins, designated A to G. BoNT/A is of particular interest, since it is frequently implicated in cases of botulism and is a significant threat in bioterrorism (1, 10).

BoNT is a 150-kDa protein composed of a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bond and noncovalent molecular interactions (24). The heavy chain (H) has two functional domains, a transmembrane domain and a receptor binding domain. The light chain (L) is a zinc-dependent protease which specifically cleaves one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors, resulting in the blockage of evoked acetylcholine release at the skeletal neuromuscular junction (8).

Previous studies have found that the bont genes of all strains of C. botulinum and neurotoxigenic strains of Clostridium butyricum and Clostridium baratii have a set of genes located upstream of the bont and ntnh genes that are organized as gene clusters (5, 7, 23). The two known primary types of clusters are (i) a hemagglutinin (ha) cluster and (ii) an orfX cluster with open reading frames (ORFs) of unknown functions. The ha cluster consists of genes encoding HA17, HA33, HA70, BotR, and NTNH. The orfX cluster consists of genes encoding ORFX3, ORFX2, ORFX1, P47, P21, and NTNH. Previous studies indicate that BoNT/A subtypes possess either a ha cluster or an orfX cluster associated with their expressed bont gene, depending on the subtype and strain (5, 11, 13-15, 33).

It has been shown that the BoNT complex can form stable toxin complexes (TCs) of various sizes, including LL-TC (~900 kDa), L-TC (~500 kDa), and M-TC (~300 kDa) composed of various combinations of HA proteins, NTNH, and BoNT (19, 21, 23, 29, 31, 34). M-TC contains BoNT and NTNH but has no HA proteins, whereas LL-TC and L-TC contain different ratios of the BoNT, NTNH, and HA proteins (21, 22, 29, 34). The biological and structural roles of the complex proteins are not completely characterized, although it has been proposed that they serve the role of protecting BoNT from harsh conditions, including pH, salt, temperature, and digestive enzymes, and that they assist BoNT translocation across the intestinal epithelial layer (2, 6, 17). A recent report indicated that the nontoxic proteins serve as adjuvants and contribute to the immunogenicity of BoNT/A (25).

The production of botulinum TCs is known to vary with different serotypes and strains, medium composition, and culture conditions (21, 24, 31). The LL-TC has only been observed in proteolytic strains (group I). Serotype A to D strains produce M-TC and L-TC in their culture medium, while serotype E and F strains produce only M-TC (17, 18).

In 1986, a Japanese group isolated four HA-negative C. botulinum strains from infant botulism cases that produced only M-TC (300 kDa). They assigned the strains to subtype A2 (14, 30). In 2004, our laboratory confirmed on a genomic level that the BoNT/A2 subtype contained the orfX cluster instead of the ha cluster (12). Since then, more arrangements and combinations of neurotoxin gene clusters were characterized along with more BoNT subtypes (13, 20, 33). However, the function of the orfX genes and the role of the presumptive protein products and their role in the TCs are still unknown, including whether ORFX proteins can form a TC with the expressed toxin analogous to the ha cluster proteins.

In this study, the BoNT/A2 TC was purified from a native culture to determine if the orfX cluster proteins remain associated with BoNT/A2. To better understand the role of the orfX cluster genes, the orfX cluster proteins of C. botulinum A2 strains (ORFX1, ORFX3, P47, and the middle part of NTNH) was expressed using either an Escherichia coli or a C. botulinum expression system in this study. Antibodies against individual expressed orfX cluster proteins were then raised by immunizing a rabbit and mice. These antibodies were then used as probes to investigate the expression pattern of the orfX cluster genes in the native A2 culture. ORFX2, which could not be expressed, was detected by N-terminal protein sequencing.


Strains, medium, cells, and culture conditions.

C. botulinum strains ATCC 3502 and Kyoto F were grown under anaerobic conditions for 96 h in toxin production medium (TPM) containing 2% NZ Case TT, 1% yeast extract, and 0.5% glucose (pH 7.4). Additional C. botulinum strains used in the study were CDC/A3 (BoNT/A3), 657 Ba (BoNT/A4), and OkraB (BoNT/B). CDC/A3 (BoNT/A3) was obtained from the Centers for Disease Control and Prevention. The genomic and BoNT/A3 profiles were analyzed using PFGE and sequencing to show that it is genetically identical to the Loch Maree strain (unpublished data). Genomic DNAs were isolated as described previously (11). E. coli strains were grown in LB medium (5 g yeast extract, 10 g Bacto tryptone, and 10 g NaCl per liter). All of the medium ingredients were purchased from Difco. TOP10 competent cells were purchased from Invitrogen, and Codon/Plus BL21 competent cells were purchased from Stratagene. RNase A, DEAE-Sephadex A-50, and CM-Sepharose (CL-6B) were purchased from Sigma. Mono-Q (HR5/5) and mono-S (HR5/5) were purchased from Pfizer-Pharmacia Inc.

Preparation of E. coli expression constructs containing partial ntnh/A1, partial ntnh/A2, and BoNT/A2-associated orfX1, orfX2, orfX3, and p47 fused to an N-terminal 6-His tag, respectively.

Initially, full-length ntnh/A1 was amplified from genomic DNA using appropriate primers from IDT (Table (Table1),1), the High Fidelity Super Mix PCR kit (Invitrogen), and the following PCR regimen: 95°C for 2 min, 25 cycles of 95°C for 1 min, an annealing step of 45 s at 44°C, and 72°C for 3 min for extension, followed by 1 cycle of extension at 72°C for 10 min. The PCR product was then cloned into the Gateway pDONOR221 plasmid via a BP reaction (Invitrogen) to create a pEntry clone. The expression vector was constructed via an LR reaction between the pEntry clone and plasmid pDest17 (Invitrogen) to express the recombinant protein in E. coli Codon/Plus cells (Stratagene). A1 ntnh consists of 3,579 bp encoding 1,193 amino acids (aa) with a molecular mass of 138 kDa. Efforts to express full-length ntnh were unsuccessful for unknown reasons. Since antibody production does not require the full-length protein, the ntnh gene was apportioned into three approximately equal-size segments that covered the full length of the gene, and these were expressed individually to enable expression. The first segment spanned 1,152 bp starting at the start codon and encoded a 44.3-kDa segment (aa 1 to 384) of NTNH; the second segment covered the middle portion of 1,365 bp encoding a 52.7-kDa segment (aa 375 to 830) of NTNH, and the third segment covered the remainder of the C-terminal 1,116 bp encoding a 44.7-kDa fragment (aa 822 to 1194) of NTNH. The primers used to amplify these regions are listed in Table Table1.1. These constructs were generated in a manner similar to the aforementioned procedure but with a shorter PCR extension time. The resulting expression vectors were designated pDest17ntnh A2 1st to 3rd, respectively. Each of the partial ntnh genes was fused to an N-terminal 6-His tag in the expression vector. The recombinant clones with the appropriate genes were verified by sequencing performed by the University of Wisconsin Biotechnology Center. The final sequencing results were analyzed using the Vector NTI Suite Program (Invitrogen).

General characteristics of genes expressed in an E. coli expression system and the primers used for amplification

A similar procedure was used to create pDest17 expression vectors for the complex protein genes described above. The primers used to amplify these genes are listed in Table Table1.1. Similar to BoNT/A1-associated NTNH, only the piece located in the middle of BoNT/A2-associated NTNH (aa 397 to 757) could be expressed in E. coli.

Purification of proteins expressed in E. coli.

The clones harboring the appropriate genes were transformed into Codon/Plus BL21 competent cells. One colony from the transformed cells was inoculated into 2 ml LB medium containing 100 μg/ml ampicillin and shaken at 175 rpm overnight at 37°C. From this culture, 500 μl was then inoculated into 50 ml LB medium containing 100 μg/ml ampicillin and shaken at 37°C until an optical density at 550 nm (OD550) of 0.5 to 0.6 was attained. Isopropyl-β-d-thiogalactopyranoside (IPTG; Invitrogen) was added at a final concentration of 1 mM to induce expression for at least 3 h. Cell pellets were collected by centrifugation at 5,000 rpm for 15 min. The protein expression levels were analyzed by Coomassie blue-stained NuPage 4 to 12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (Invitrogen). The protein solubilities were analyzed and the expressed proteins were purified by following the manufacture's protocol for the Probond Protein Purification System kit from Invitrogen.

Generation of a C. botulinum expression construct containing BoNT/A2-associated orfX1 with a C-terminal 6-His tag.

The effort to express ORFX1 in an E. coli expression system was unsuccessful. A C. botulinum expression system was then used to express ORFX1 instead. The full-length orfX1 gene with the restriction sites SacI on the 5′ end and SphI on the 3′ end was first PCR amplified from the construct pDest17orfX1 containing the orfX1 gene as described above. The primers used to amplify DNA were orfX1 forward C.B (5′-AGAGAGCTCGATGAATCAAACATTTTCTTTTAATTTTG-3′) and orfX1 reverse C.B (5′-AGAGCATGCTGCGATTTGCAATAAATT-3′; underlining indicates a SacI or SphI site, and bold indicates the orfX1 start codon). The PCR product was then cloned into the pGEMT vector (Promega) and verified by DNA sequencing. The recombinant plasmid DNA was double digested with SacI and SphI, and the fragment containing orfX1 was subcloned into the clostridial expression vector pMVP410 (27) also double digested with SacI and SphI. The resulting construct, containing the orfX1 gene fused with a C-terminal 6-His tag, was designated pMVP442. The clones containing the orfX1 gene were verified by restriction enzyme digestion and DNA sequencing.

Purification of ORFX1 expressed in C. botulinum.

The positive recombinant plasmid pMVP442 DNA was first transformed into E. coli donor strain CA434. Then, pMVP442 was transferred into nontoxigenic C. botulinum type A transposon Tn916 mutant strain LNT01, which lacks the entire toxin gene cluster, by conjugation (4). ORFX1 was expressed and extracted by the methods previously described (27). The expression of ORFX1 was analyzed with anti-His antibody by Western blot analysis. The protein was then purified by using the Probond protein purification system under denaturing conditions. The purified protein was analyzed by Coomassie blue-stained NuPage 4 to 12% Bis-Tris SDS-PAGE.

Generation of polyclonal antibodies by immunizing a rabbit and mice.

A New Zealand White rabbit (Harlan Sprague-Dawley Inc.) was immunized with 500 μg of the recombinant second segment of the BoNT/A2-associated NTNH protein (aa 397 to 757). Female ICR mice (Harlan Sprague-Dawley Inc.) were immunized with 10 μg each of ORFX1, ORFX3, or P47 from A2 or the BoNT/A1-associated second segment of the NTNH protein (aa 375 to 830). Two mice were immunized for each protein. The proteins were mixed with aluminum hydroxide adjuvant at a 1:1 ratio for 20 min at room temperature before injection. Both the rabbit and mice were boosted twice at 2-week intervals. The titer was analyzed 2 weeks after the final boost. Immunized mice were bled from the tail vein, and the immunized rabbit was bled from the ear vein. The serum containing antibodies against specific proteins was used directly for Western blot analysis. All animal work was done using IACUC-approved protocols in AALAC-accredited facilities.

Purification of BoNT/A2 TC and BoNT/A2.

The BoNT/A2 TC was purified to determine the proteins that remain associated with the BoNT. The purification procedures were based on those used for BoNT/A1 (9) and E (16), with some modifications. A carboy with 10 liters of sterile TPM was inoculated with 10 ml of actively growing Kyoto F culture (24 h) and incubated statically for 96 h at 37°C. The culture was cooled for 60 min on ice, and the pH was lowered to 3.5 by addition of 3 N H2SO4. The precipitate that formed was collected by centrifugation and washed for 5 h with distilled water. The pellet was extracted twice by suspension in 0.1 M sodium citrate buffer (pH 5.5) with gentle stirring for 2 h at room temperature. The extracted supernatants were combined. Ammonium sulfate was then added to the extract supernatants to 60% saturation (39 g/100 ml), and the mixture was kept at 4°C overnight. The precipitate was collected by centrifugation and resuspended in 50 mM NaPO4 buffer (pH 6.4). RNase A was added at a concentration of 100 μg/ml, and the mixture was incubated for 5 h at 37°C. The solution after digestion was centrifuged to remove insoluble material, and the protein was then precipitated with solid ammonium sulfate (39 g/100 ml). The crude toxin extract was obtained at this step. An aliquot of this crude toxin extract was used to perform N-terminal protein sequencing to determine if ORFX1 or ORFX2 was present. The C. botulinum A2 crude toxin extract was separated by NuPage 4 to 12% Bis-Tris SDS-PAGE, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen). The membrane was stained using Coomassie blue, and the targeted bands were sequenced by the facilities at the Medical College of Wisconsin (32).

Chromatography steps used to obtain the purified TC and neurotoxin. (i) DEAE chromatography at pH 5.5.

The ammonium sulfate precipitate pellet was collected by centrifugation, resuspended in 0.05 M Na citrate (pH 5.5), and dialyzed for 4 h at room temperature with three dialysis changes at 1-h intervals. The dialyzed solution was centrifuged to remove insoluble material, and the supernatant was loaded onto a DEAE-Sephadex A-50 column equilibrated with 50 mM Na citrate buffer (pH 5.5) at room temperature. Fractions were monitored at OD278 and analyzed by SDS-PAGE. The fractions containing the crude TC were pooled and precipitated with solid ammonium sulfate (39 g/100 ml) and stored at 4°C.

(ii) CM-Sepharose chromatography.

The ammonium sulfate-precipitated crude TC from the DEAE (pH 5.5) column was collected by centrifugation and resuspended in 0.025 M sodium citrate buffer (pH 6.0). The solution was dialyzed at room temperature for 6 h and loaded onto a CM-Sepharose column. The column was washed with 25 mM Na citrate buffer (pH 6.0), and fractions were monitored at OD278 and analyzed by SDS-PAGE. The fractions containing pure TC were pooled and precipitated by addition of ammonium sulfate (39 g/100 ml).

(iii) FPLC mono-Q chromatography.

The precipitated TC from the CM-Sepharose chromatography was collected by centrifugation, resuspended in 20 mM sodium phosphate buffer (pH 8.0), and dialyzed for 4 h at room temperature. The dialyzed solution was loaded onto a fast protein liquid chromatography (FPLC) mono-Q column (1 ml/min) for separation of the toxin from NTNH. A 0 to 0.35 M NaCl gradient was applied to the column to elute the bound material. The A2 toxin, along with several minor contaminating proteins, was recovered in the first peak. Those fractions were pooled and precipitated with ammonium sulfate.

(iv) FPLC mono-S chromatography.

The precipitated pellet obtained by mono-Q chromatography was collected by centrifugation, resuspended in 20 mM sodium phosphate buffer (pH 7.0), and dialyzed for 4 h at room temperature before being loaded onto an FPLC mono-S column and eluted at a 1-ml/min flow rate. The fractions were monitored at OD278, and the column was washed until the OD278 reached background levels. The toxin was then eluted with 20 mM NaPO4 buffer containing 0.5 M NaCl and analyzed for purity by 4 to 12% SDS-PAGE. The purity of the A2 toxin recovered by mono-S chromatography was estimated to be >95% based on SDS-PAGE results.

The specific toxicity of the purified A2 toxin was determined by intraperitoneal injection of the following seven different toxin amounts into groups of mice (four mice per group): 15, 10, 6.67, 4.45, 2.97, and 1.98 pg/mouse. The toxin was diluted in 0.5 ml gel phosphate buffer and injected intraperitoneally. The injected mice were observed for 4 days. The 50% lethal dose (LD50)/mg of toxin was calculated by the method of Reed and Muench (28).

Analysis of subtype-specific NTNH immunoreactivity.

C. botulinum strains comprising subtypes A1, A2, A3, A4, and B were evaluated. Individual strains were inoculated into 10 ml TPGY medium (5) and grown at 37°C for 4 days to promote cellular lysis and release of the neurotoxin. The medium was then centrifuged, and the supernatant was used for the Western blot analysis. Antisera against BoNT/A1-associated NTNH (aa 375 to 830) and BoNT/A2-associated NTNH (aa 397 to 757) were used as probes.


Expression of NTNH/A1- and BoNT/A2-associated neurotoxin gene cluster proteins.

Genes and gene fragments including ntnh/A1 and three ntnh/A1 segments, BoNT/A2-associated full-length ntnh, and BoNT/A2-associated orfX1, orfX2, orfX3, and p47, were cloned into the Invitrogen Gateway E. coli expression system. Associated proteins were expressed under the control of the T7 promoter (see Fig. S1 in the supplemental material). P47 and ORFX3 were successfully expressed using the E. coli expression system (see Fig. S2 in the supplemental material). Full-length NTNH/A1 and NTNH/A2 were not successfully expressed. This led to attempts to express parts of ntnh by dividing the gene into three approximately equal-size segments which covered the full length of the gene (Table (Table11 shows the segment details). Only the middle segment of each, comprising a fragment of 52.7 kDa of NTNH/A1 and a fragment of 46.6 kDa of NTNH/A2, was successfully expressed (see Fig. S2 in the supplemental material).

While the orfX1 and orfX2 genes were successfully cloned into the expression vectors, attempts to induce their expression were unsuccessful, as their expression appeared to induce cell lysis and death. The results indicated that both ORFX1 and ORFX2 are toxic to E. coli (see Fig. S3 in the supplemental material). An alternative strategy to identify ORFX1 and ORFX2 in the crude C. botulinum A2 toxin extract was used. The crude toxin extract was separated by SDS-PAGE, and bands corresponding to the predicted molecular weights of ORFX1 and ORFX2 were sequenced. The N-terminal sequencing result was Asn-Asn-Leu-Lys-Pro-Phe-Ile-Tyr-Tyr-Asp and confirmed the presence of ORFX2 in the crude extract, but ORFX1 was not indentified (Fig. (Fig.1).1). Since ORFX1 could not be identified by N-terminal sequencing in the extract, an attempt was made to express ORFX1 using a C. botulinum expression system instead of an E. coli expression system (see Fig. S1 in the supplemental material). The results showed that ORFX1 was successfully expressed in C. botulinum LNT01 (see Fig. S2 in the supplemental material). All of the expressed proteins (second segment of NTNH/A1, second segment of NTNH/A2, P47, ORFX1, and ORFX3) were purified by affinity chromatography using Ni+ columns under denaturing conditions. The purity of the proteins was confirmed by SDS-PAGE analysis (see Fig. S4 in the supplemental material).

FIG. 1.
Protein sequencing of bands from BoNT/A2 crude extract. The A2 crude toxin extract was separated by NuPage 4 to 12% Bis-Tris SDS-PAGE, transferred onto a PVDF membrane, and then stained with Coomassie blue. A few bands were excised for protein ...

Generation of antisera against individual recombinant cluster proteins in either a rabbit or mice.

Antibodies against the internal segment of NTNH/A2 (aa 397 to 757) were raised in a rabbit. Antibodies against ORFX1, ORFX3, P47, and the middle piece of NTNH (aa 375 to 830) associated with BoNT/A1 were raised separately in mice.

Western blot analyses of the antisera against the individual expressed recombinant neurotoxin cluster proteins.

To verify the ability of each antiserum to recognize its expressed recombinant protein, Western blot analyses were performed. Recombinant proteins were induced by 1 mM IPTG in Codon/Plus BL21 cells or in C. botulinum LNT01 cells containing the recombinant plasmids, respectively. Cell pellets were collected and used for SDS-PAGE and Western blot analysis. Each of the recombinant proteins was detected at the expected size using the antiserum that was generated against the specific protein (Fig. (Fig.2).2). The data indicated that each antiserum generated could recognize its target expressed protein.

FIG. 2.
Western blot analysis of expressed neurotoxin cluster proteins using protein-specific antiserum. Data showed that each antiserum recognizes its specific protein expressed in an E. coli or C. botulinum expression system. (Lanes 1 to 3 and 5) E. coli Codon/Plus ...

Detection of A2 neurotoxin gene cluster proteins in crude BoNT/A2 extract.

Western blot analysis was performed with crude A2 toxin extract (Fig. (Fig.3)3) to determine if the neurotoxin cluster proteins were expressed in the native A2 culture. Specific antisera were used to probe for each protein in the cluster. NTNH, P47, and ORFX3 were detected in the crude extract by Western blot analysis (Fig. (Fig.3).3). ORFX1 was not detected in the crude extract, although the antiserum was able to detect recombinant ORFX1 in independent Western blot analyses. ORFX2 was detected by N-terminal protein sequencing following SDS-PAGE (Fig. (Fig.1).1). The data indicate that all of the neurotoxin cluster proteins were detected in the native culture, except ORFX1.

FIG. 3.
Western blot analyses of the neurotoxin cluster proteins of A2 and NTNH of A1 in crude toxin extract using antisera against specific proteins. The crude toxin extract was made from either A1 or A2 culture. Data showed that BoNT/A2-associated NTNH, ORFX3, ...

Purification and characterization of the BoNT/A2 complex and neurotoxin.

The TC and BoNT/A2 were purified to determine the possible presence of TC proteins associated with BoNT/A2. SDS-PAGE data indicated that the A2 TC was composed of two different proteins, BoNT/A2 and NTNH (Fig. (Fig.4A).4A). BoNT/A2 was confirmed by SDS-PAGE under reducing condition and mouse bioassay (Fig. (Fig.4A);4A); NTNH was confirmed by Western blot analysis (Fig. (Fig.4B)4B) using NTNH-specific antiserum. BoNT/A2 was separated from NTNH by additional chromatography steps. SDS-PAGE data showed that ≥95% pure BoNT/A2 was obtained after the final chromatography step (Fig. (Fig.4C).4C). The specific toxicity of the 150-kDa protein was determined to be ~4 × 108 LD50/mg.

FIG. 4.
SDS-PAGE and Western blot analysis of purified A2 TC and BoNT/A2. (A) Coomassie blue-stained SDS-PAGE of the A2 TC. A2 TC proteins were separated by 4 to 12% Bis-Tris SDS-PAGE under reducing and nonreducing conditions. (B) Western blot analysis ...

Analysis of subtype-specific NTNH immunoreactivity.

To examine if NTNH has cluster-specific immunoreactivity, we performed a Western blot analysis comparing strains of subtypes containing different types of neurotoxin cluster genes. C. botulinum strains comprising subtypes A1, A2, A3, A4, and B were evaluated. BoNT/A1 was present in a ha cluster. BoNT/A2 and BoNT/A3 are associated with orfX clusters, and subtype A4 possesses two sets of the BoNT-related clusters, BoNT/B associated with a ha cluster and BoNT/A4 associated with an orfX cluster. Antisera against either BoNT/A1-associated NTNH (aa 375 to 830) and BoNT/A2-associated NTNH (aa 397 to 757) were used as probes. The antiserum against BoNT/A1-associated NTNH only recognized NTNH associated with the ha cluster, which is found in subtypes A1, A4 (BoNT/B), and B1. Antiserum to BoNT/A2-associated NTNH only recognized NTNH proteins associated with the orfX cluster, which is found in subtypes A2, A3, and A4 (BoNT/A4) (Fig. (Fig.5).5). The intensity of the NTNH detected by the anti NTNH/A2 antisera from the cultures of the A3 and A4 strains was much weaker than that from the A2 strain (Fig. (Fig.5B).5B). We think the reason is that the yield of BoNT/A3 or BoNT/A4 is much lower (approximately 1,000-fold) than that of BoNT/A2. In addition, data showed that A4 strains not only contain two different sets of neurotoxin clusters but also produce two different kinds of NTNH, BoNT/B-associated NTNH and BoNT/A4-associated NTNH. However, the BoNT/B-associated NTNH protein from the ha cluster is produced at much higher levels than the BoNT/A4-associated NTNH protein from the orfX cluster (Fig. (Fig.5A,5A, A4 lane, and B, A4 lane). The antisera from the two different types of clusters did not cross-react with each other. These data indicated that the NTNH proteins from different subtypes have specific immunoreactivities.

FIG. 5.
Western blot analysis of subtype-specific NTNH immunoreactivity. C. botulinum A1, A2, A3, A4, and B strains were selected to test NTNH-specific immunoreactivity. Supernatants from a culture of each strain were collected and separated by NuPage 4 to 12% ...


In this study, we have purified the TC and BoNT in an A2 strain previously associated with infant botulism. As described below, the complex was composed of NTNH and BoNT only and did not possess ORFX proteins. The purified neurotoxin consisted of a dichain 150-kDa protein with a specific toxicity of ~4 × 108 LD50/mg, which is slightly higher than that reported for certain other BoNTs (34).

In C. botulinum strains with a ha cluster, it has been shown that the BoNT can form various sizes of TCs with NTNH and HA proteins, depending on the serotype and strain (31). It has been postulated that the TCs can protect BoNT from digestive enzymes and conditions present in the gastrointestinal tract, including pH and temperature, and assist the BoNT in translocation across the intestinal mucosal layer. In C. botulinum strains with an orfX cluster, it is unknown whether orfX cluster proteins are expressed in the native culture or if they participate in the formation of a TC. To better understand the association of BoNT with the orfX cluster, a C. botulinum A2 strain (Kyoto F) possessing a neurotoxin orfX cluster was selected for study. The orfX cluster proteins (ORFX1, ORFX3, P47, and part of NTNH) of the C. botulinum A2 strain was expressed by using either an E. coli or a C. botulinum expression system. Full-length ntnh from A1 or A2 was not successfully expressed. It is possible that the N terminus of NTNH was cleaved after expression. This hypothesis was supported by the recent report that focused on a study of TC in a D strain (26). The data we obtained in our laboratory also showed that NTNH was not stable by itself; pure NTNH degraded much faster than NTNH associated with toxin (see Fig. S5A in the supplemental material). Our data demonstrated that all of the neurotoxin cluster proteins except ORFX1 were present in the A2 culture. ORFX1 and ORFX2 were not detected in the E. coli host cells. The E. coli cells started to lyse when ORFX1 or ORFX2 expression was induced, suggesting that ORFX1 and ORFX2 were toxic to the E. coli cells. In a C. botulinum expression system, ORFX1 was expressed at a low level.

Antibodies against the individual orfX cluster proteins were developed by immunizing a rabbit or mice. These antibodies were used as probes to determine if the A2 orfX cluster genes were expressed in the native A2 culture. We could not detect the ORFX1 protein in the A2 crude extract either by using the antiserum against recombinant ORFX1 or by protein sequencing of candidate bands of ~16.5 kDa by SDS-PAGE. ORFX1 did not show sequence similarity to any known proteins in a blast search. We postulated that either ORFX1 is not translated into a protein in the A2 strain or its level of expression is very low compared to that of the other cluster genes, such that it could not be detected. ORFX2 was detected by protein sequencing of bands from the BoNT/A2 crude extract separated by SDS-PAGE. P47 and ORFX3 were also detected in the A2 culture by Western blot analysis. Western blot assay results also showed bands in addition to ORFX3 or NTNH when ORFX3 or NTNH antibodies were used as probes. These bands appear to be truncated forms of NTNH or ORFX3. Truncated complex proteins were also observed in a previous study (6).

Our data indicated that the C. botulinum A2 strain forms a TC composed of BoNT/A2 and NTNH but lacking the other orfX cluster proteins. This suggested that orfX cluster proteins, which are transcribed in the opposite direction of the bont and ntnh genes, are not part of the TC. The P47, ORFX2, and ORFX3 proteins are present in the native culture; however, their roles in the cells are unknown. A previous study indicated that bont/a2, ntnh, and p47 were expressed on the same tricistronic transcript (12). The genetic relationship of the orfX orfX2, orfX3, and p47 cluster genes is unknown. When the nucleotide and amino acid sequences of these genes are compared, they exhibit very little homology, less than 20% identity and similarity. However, when the ORFX2 and ORFX3 sequences were analyzed using the NCBI blast search engine, a similarity of secondary structure to P47 was observed. Specifically, a P47 subfamily motif was identified in ORFX2 at aa 174 to 433 and in ORFX3 at aa 5 to 287 and had related secondary structures. It is intriguing that ORFX2 and ORFX3 have secondary structure similarities to P47, but the function of these proteins is unknown.

An intriguing observation of this study was that analysis of NTNH by Western blotting demonstrated cluster-specific (HA or OrfX) immunoreactivity. Previous data showed that NTNH associated with different neurotoxin cluster types had a similarity of around 80% and an identity of around 75% across the entire length of the protein (20). The internal ~50-kDa regions of NTNH/A1 and NTNH/A2 used in this study to create antibodies had 80.4% similarity and 74.4% identity. However, antibodies prepared against the internal regions of NTNH/A1 or NTNH/A2 were cluster specific in their reactions tested by Western blotting. Because of the low production of BoNT/A3 and NTNH, we detected a very weak NTNH band from the supernatant of a 4-day-old culture of the A3 strain. A much stronger NTNH band was detected from the purified BoNT/A3 TC (see Fig. S5B in the supplemental material). The antibodies produced in this study for the cluster-specific NTNH proteins can be used as a tool to identify the expression of different clusters within BoNT-producing bacteria. The antibodies will be especially useful for bivalent strains in identifying the expressed specific cluster type associated with the toxin.

In conclusion, our laboratory has revealed interesting differences between ha and orfX clusters. Specifically, the ORFX proteins are not components of the A2 complex, and the internal region of NTNH proteins associated with ha or orfX clusters have distinct antibody recognition. Further studies of cluster gene expression, protein-protein interactions, and determination of the structure of BoNT complexes are needed to better understand the role of toxin-associated proteins in C. botulinum.

Supplementary Material

[Supplemental material]


This work was sponsored by the NIH/NIAID Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE) program. We acknowledge membership in the Pacific Southwest Regional Center of Excellence and support from grant U54 AI065359 and from the Region V Great Lakes RCE (NIH award 1-U54-AI-057153).


[down-pointing small open triangle]Published ahead of print on 13 November 2009.

Supplemental material for this article may be found at


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