Substrate cleavage and duration of action of botulinum neurotoxin type FA ("H, HA").

Botulinum neurotoxin (BoNT) type FA is the only known naturally occurring chimeric BoNT of domains of BoNT/A and BoNT/F. BoNT/FA consists of an F5-like light chain (LC), a unique heavy chain (HC) translocation domain, and a HC receptor binding domain similar to BoNT/A1. Previous analyses of purified BoNT/FA have indicated a 5-10-fold greater potency in cultured human or rat neurons as compared to BoNT/A1 and a 400-500-fold greater potency compared to BoNT/B1. However, in vivo potency in mice was about 5-fold lower than BoNT/A1 or/B1. In this report, species specificity was examined by cell-based assays using primary neurons from mice and examining VAMP1 and 2 cleavage. The data indicated similar potency of BoNT/FA in primary mouse spinal cord neurons as previously observed in primary rat and human induced pluripotent stem cell (hiPSC) derived neuronal cell models, and equal enzymatic cleavage of mouse VAMP1 and 2 isoforms. Since the duration of action of BoNTs is due to continuous enzymatic activity of the LC in the neuronal cytosol, BoNT/FA was expected to have a short duration of action due to its F-type LC. In this report the duration of action of BoNT/FA was compared to that of BoNT/F1,/F5, and/B1 in both hiPSC derived neurons and in the in vivo mouse model. The data indicate a duration of action of BoNT/FA similar to BoNT/B1, while BoNT/F5 had a short duration of action similar to BoNT/F1.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium.IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1,000-fold and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation of growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples and into the enigmatic function of BoNTs in nature.

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

Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 10(7) mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a "treatment-resistant" and highly potent toxin. However, the toxin's chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

Remote Axial Tuning in Microscopy Utilizing Hydrogel-Driven Tunable Liquid Lens.

This work presents the use of a tunable-focus thermo-responsive hydrogel based liquid lens in combination with an objective lens to achieve remote axial focusing in a conventional microscopy. The goal of this design is to eliminate image distortion due to sample vibrations caused by mechanical stage scanning. This approach reduces the mechanical complexity and power consumption due to the use of electrically tunable lenses while achieving a two-fold increase in the axial scanning range. The merits of the proposed design were demonstrated by characterizing a customized microscope system over a scanning range of 1700 µm. A lateral resolution of 2 µm was obtained consistently throughout the scanning range. Healthy Spodoptera frugiperda Sf21 insect cells imaging was used to verify the depth scanning ability and the resolution of our remote focusing microscope system.

Prevalence of toxin-producing Clostridium botulinum associated with the macroalga Cladophora in three Great Lakes: growth and management.

The reemergence of avian botulism caused by Clostridium botulinum type E has been observed across the Great Lakes in recent years. Evidence suggests an association between the nuisance algae, Cladophora spp., and C. botulinum in nearshore areas of the Great Lakes. However, the nature of the association between Cladophora and C. botulinum is not fully understood due, in part, to the complex food web interactions in this disease etiology. In this study, we extensively evaluated their association by quantitatively examining population size and serotypes of C. botulinum in algal mats collected from wide geographic areas in lakes Michigan, Ontario, and Erie in 2011-2012 and comparing them with frequencies in other matrices such as sand and water. A high prevalence (96%) of C. botulinum type E was observed in Cladophora mats collected from shorelines of the Great Lakes in 2012. Among the algae samples containing detectable C. botulinum, the population size of C. Botulinum type E was 10(0)-10(4) MPN/g dried algae, which was much greater (up to 10(3) fold) than that found in sand or the water column, indicating that Cladophora mats are sources of this pathogen. Mouse toxinantitoxin bioassays confirmed that the putative C. botulinum belonged to the type E serotype. Steam treatment was effective in reducing or eliminating C. botulinum type E viable cells in Cladophora mats, thereby breaking the potential transmission route of toxin up to the food chain. Consequently, our data suggest that steam treatment incorporated with a beach cleaning machine may be an effective treatment of Cladophora-borne C. botulinum and may reduce bird mortality and human health risks.

Immunoprecipitation of native botulinum neurotoxin complexes from Clostridium botulinum subtype A strains.

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.

Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro.

Botulinum neurotoxins (BoNTs) are synthesized by Clostridium botulinum and exist as seven immunologically distinct serotypes designated A through G. For most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. BoNT/A1 is the most well-characterized subtype of the BoNT/A serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. In contrast, much remains unknown of the other BoNT/A subtypes. In this study, BoNT/A subtype 1 (BoNT/A1) to BoNT/A5 were characterized utilizing a mouse bioassay, an in vitro cleavage assay, and several neuronal cell-based assays. The data indicate that BoNT/A1 to -5 have distinct in vitro and in vivo toxicological properties and that, unlike those for BoNT/A1, the neuronal and mouse results for BoNT/A2 to -5 do not correlate with their enzymatic activity. These results indicate that BoNT/A1 to -5 have distinct characteristics, which are of importance for a greater understanding of botulism and for pharmaceutical applications.

Association of toxin-producing Clostridium botulinum with the macroalga Cladophora in the Great Lakes.

Avian botulism, a paralytic disease of birds, often occurs on a yearly cycle and is increasingly becoming more common in the Great Lakes. Outbreaks are caused by bird ingestion of neurotoxins produced by Clostridium botulinum, a spore-forming, gram-positive, anaerobe. The nuisance, macrophytic, green alga Cladophora (Chlorophyta; mostly Cladophora glomerata L.) is a potential habitat for the growth of C. botulinum. A high incidence of botulism in shoreline birds at Sleeping Bear Dunes National Lakeshore (SLBE) in Lake Michigan coincides with increasingly massive accumulations of Cladophora in nearshore waters. In this study, free-floating algal mats were collected from SLBE and other shorelines of the Great Lakes between June and October 2011. The abundance of C. botulinum in algal mats was quantified and the type of botulism neurotoxin (bont) genes associated with this organism were determined by using most-probable-number PCR (MPN-PCR) and five distinct bont gene-specific primers (A, B, C, E, and F). The MPN-PCR results showed that 16 of 22 (73%) algal mats from the SLBE and 23 of 31(74%) algal mats from other shorelines of the Great Lakes contained the bont type E (bont/E) gene. C. botulinum was present up to 15000 MPN per gram dried algae based on gene copies of bont/E. In addition, genes for bont/A and bont/B, which are commonly associated with human diseases, were detected in a few algal samples. Moreover, C. botulinum was present as vegetative cells rather than as dormant spores in Cladophora mats. Mouse toxin assays done using supernatants from enrichment of Cladophora containing high densities of C. botulinum (>1000 MPN/g dried algae) showed that Cladophora-borne C. botulinum were toxin-producing species (BoNT/E). Our results indicate that Cladophora provides a habitat for C. botulinum, warranting additional studies to better understand the relationship between this bacterium and the alga, and how this interaction potentially contributes to botulism outbreaks in birds.

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).

Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 µL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.

Genetic manipulation of Xanthophyllomyces dendrorhous and Phaffia rhodozyma.

The yeasts Xanthophyllomyces dendrorhous (teleomorph) and Phaffia rhodozyma (anamorph) are of basidiomycetous affinity and have the unique property among yeasts of producing the carotenoid pigment astaxanthin. Astaxanthin imparts the attractive coloration to salmonids, crustaceans, and several birds such as the flamingo, and it has considerable economic value. Microbiological and genetic techniques for manipulation are rudimentary in the yeast, while their utility would be valuable for strain development including hypermutants that overproduce astaxanthin. Here we describe methods for manipulation of the yeast, including induction of the sexual stage with basidiospore formation, methods for isolation of mutants (particularly mutants affected in carotenoid biosynthesis) as well as techniques for isolation and analysis of carotenoids. These methods are valuable for understanding the biology and enhancing the biotechnology value of the yeast.

Identification of amino acids essential for estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1.

As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.

Purification and characterization of a novel subtype a3 botulinum neurotoxin.

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are of considerable importance due to their being the cause of human and animal botulism, their potential as bioterrorism agents, and their utility as important pharmaceuticals. Type A is prominent due to its high toxicity and long duration of action. Five subtypes of type A BoNT are currently recognized; BoNT/A1, -/A2, and -/A5 have been purified, and their properties have been studied. BoNT/A3 is intriguing because it is not effectively neutralized by polyclonal anti-BoNT/A1 antibodies, and thus, it may potentially replace BoNT/A1 for patients who have become refractive to treatment with BoNT/A1 due to antibody formation or other modes of resistance. Purification of BoNT/A3 has been challenging because of its low levels of production in culture and the need for innovative purification procedures. In this study, modified Mueller-Miller medium was used in place of traditional toxin production medium (TPM) to culture C. botulinum A3 (CDC strain) and boost toxin production. BoNT/A3 titers were at least 10-fold higher than those produced in TPM. A purification method was developed to obtain greater than 95% pure BoNT/A3. The specific toxicity of BoNT/A3 as determined by mouse bioassay was 5.8 × 10(7) 50% lethal doses (LD(50))/mg. Neutralization of BoNT/A3 toxicity by a polyclonal anti-BoNT/A1 antibody was approximately 10-fold less than the neutralization of BoNT/A1 toxicity. In addition, differences in symptoms were observed between mice that were injected with BoNT/A3 and those that were injected with BoNT/A1. These results indicate that BoNT/A3 has novel biochemical and pharmacological properties compared to those of other subtype A toxins.

Purification, modeling, and analysis of botulinum neurotoxin subtype A5 (BoNT/A5) from Clostridium botulinum strain A661222.

A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences--specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.

Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1.

Botulinum neurotoxins (BoNTs), the causative agent of human botulism, are the most potent naturally occurring toxins known. BoNT/A1, the most studied BoNT, is also used as an important biopharmaceutical. In this study, the biological activity of BoNT/A1 is compared to that of BoNT/A2 using neuronal cell models. The data obtained indicate faster and increased intoxication of neuronal cells by BoNT/A2 than BoNT/A1, and that the mechanism underlying this increased toxicity is faster and more efficient cell entry that is independent of ganglioside binding. These results have important implications for the development of new BoNT based therapeutics and BoNT countermeasures.

Synaptotagmin II peptide-bead conjugate for botulinum toxin enrichment and detection in microchannels.

This paper reports an enrichment platform for botulinum neurotoxin type B (BoNT/B) that has been realized through the fusion of bioconjugation chemistry and microfluidics. Micrometer-sized magnetic beads were conjugated to a 22mer synthetic peptide derived from the synaptotagmin II (Syt II) neuronal protein that is specific for BoNT/B binding. Exposure to BoNT/B in buffer, whole milk and fruit juices resulted in toxin capture, which was confirmed using immunofluorescence. Peptide-modified beads were integrated into arrayed, polymeric microfluidic channels, and all assay steps, from capture to detection, were performed directly in the microchannels, thereby simplifying assay utility and increasing throughput relative to existing detection methodologies. Our sensitive microscale approach required only 7 µL of intentionally adulterated sample without any pre-processing (i.e. dilution, centrifugation, filtering), and with a "hands-on" time of only 1 h to detect 16.6 pg of BoNT/B in whole milk.

SV2 mediates entry of tetanus neurotoxin into central neurons.

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.

Bleomycin induces upregulation of lysyl oxidase in cultured human fetal lung fibroblasts.

AIM: To investigate the mechanism of bleomycin (BLM)-induced pulmonary fibrosis. METHODS: Cultured human fetal lung fibroblast (HLF) cells were exposed to bleomycin (BLM) at 0-30 microg/mL for 24 h. Western blot analysis was used to detect lysyl oxidase (LO) protein expression. Real-time RT-PCR was used to detect LO mRNA level. LO catalytic activity was measured using diaminopentane as a substrate and Amplex red as a hydrogen peroxide probe. Copper (Cu) concentration was detected by flame atomic absorption spectrophotometry. RESULTS: Exposure of HLF cells to BLM at 10 microg/mL and 30 microg/mL increased LO catalytic activity to 130% and 158% of the control in the conditioned media. The expression of LO mRNA was increased to 5.5-fold of the control in HLF cells exposure to BLM at 3 microg/mL. BLM at 3 microg/mL also increased the expression of 46 kDa preproLO, 50 kDa proLO and 32 kDa mature LO to 219%, 130%, and 135% of the control, respectively. The Cu concentrations in conditioned media of cultured HLF cells exposed to BLM (10 and 30 microg/mL) were increased significantly to 1.48 and 2.46-fold of the control, respectively. CONCLUSION: Bleomycin induces upregulation of LO in cultured human fetal lung fibroblasts, which may be the mechanism of bleomycin-induced pulmonary fibrosis.

Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

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.

Phylogenetic analysis of Clostridium botulinum type A by multi-locus sequence typing.

The genus Clostridium comprises a heterogeneous group of organisms for which the phylogeny and evolutionary relationships are poorly understood. The elucidation of these evolutionary relationships necessitates the use of experimental methods that can distinguish Clostridium lineages that are time and cost effective, and can be accurately and reproducibly employed in different laboratories. Multi-locus sequence typing (MLST) has been successfully used as a reproducible and discriminating system in the study of eukaryotic and prokaryotic evolutionary biology, and for strain typing of various bacteria. In this study, MLST was applied to evaluate the evolutionary lineages in the serotype A group of Clostridium botulinum. C. botulinum type A has recently been shown to produce multiple subtypes, suggesting that it is not monophyletic as previously reported, but comprises distinct lineages. For MLST analysis, we initially evaluated 14 housekeeping genes (gapdh, tuf, sod, oppB, hsp60, dnaE, aroE, pta, 23S rDNA, aceK, rpoB, 16S rDNA, mdh and recA) for amplification and sequence analysis. In the first phase of the analysis, 30 C. botulinum type A strains producing botulinum neurotoxin subtypes A1-A4 were examined. Results of this pilot study suggested that seven of the genes (mdh, aceK, rpoB, aroE, hsp60, oppB and recA) could be used for elucidation of evolutionary lineages and strain typing. These seven housekeeping genes were successfully applied for the elucidation of lineages for 73 C. botulinum type A strains, which resulted in 24 distinct sequence types. This strategy should be applicable to phylogenetic studies and typing of other C. botulinum serotypes and Clostridium species.

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes.

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.