Cell Host & Microbe
Previews Botulinum Neurotoxins: Still a Privilege of Clostridia? Michel R. Popoff1,* 1Bacterial Toxins, Institut Pasteur, Paris, France *Correspondence:
[email protected] https://doi.org/10.1016/j.chom.2018.01.014
Botulinum neurotoxins (BoNTs) are potent bacterial toxins mostly produced by genetically diverse clostridial strains. Recently, BoNT variants have been reported in non-clostridial strains. In this issue of Cell Host & Microbe, Zhang et al. (2018) describe a novel BoNT in Entecoccus faecium. Botulinum neurotoxins (BoNTs) are bacterial protein toxins that preferentially target motor neurons and inhibit the release of acetylcholine, leading to neuromuscular blockade. BoNTs are responsible for botulism, which is a severe disease that is often fatal without treatment. Botulism is characterized by symmetrical descending flaccid paralysis of voluntary muscles progressing to respiratory failure/arrest in the severe cases as well as by inhibition of secretions (hyposalivation/xerostomia, xerophtalmia, anhydrosis, and constipation). The most common form of botulism in adults is the foodborne botulism, which results from ingestion of preformed BoNT in preserved food, whereas infant botulism consists of intestinal colonization by BoNT-producing bacterial strain and production of BoNT in situ. BoNTs are the most potent among bacteria, animal, and plant toxins as monitored by the lethal activity in experimental animals. Indeed, the lethal dose of BoNT/A is 0.1–1 mg by parental route and 70–100 mg by oral route in a human adult. The extreme potency of BoNTs supposes that these toxins might be potential highly dangerous bioweapons. Multiple scenarios of massive intoxication with BoNT/A have been suggested based on the low amount of toxin disseminated in food or water, which can theoretically kill numerous people. Therefore, BoNTs are classified in the category A of the bioterrorism agents by the United States Centers for Disease Control and Prevention. In contrast, BoNTs are also effective therapeutics widely used for the treatment of many diseases resulting from hypercholinergic activity, such as strabismus and neurologic movement disorders, and also a variety of ophthal-
mologic, gastrointestinal, urologic, orthopedic, dermatologic, dental, secretory, painful, and cosmetic indications (Jankovic, 2017). BoNTs form a large family of diverse neurotoxins divided into 8 types (A to H) and more than 40 subtypes. Each BoNT type is based on neutralization by specific polyclonal antibodies. Polyclonal antibodies raised against one BoNT type do not neutralize the other toxin types. Each BoNT type contains an increasing number of subtypes according to sequence variations (Peck et al., 2017). However, all BoNT types and subtypes retain a similar global structure and mode of action. BoNTs are produced as single-chain polypeptide of about 150 kDa, which are proteolytically activated yielding a heavy chain (Hc) and light chain (Lc) linked by a disulfide bond. Hc recognizes double cell surface receptors (ganglioside and membrane glycoprotein) and drives Lc entry into target neurons. Lc cleaves one of the three SNARE proteins (VAMP/synaptobrevin, SNAP25, and syntaxin), resulting in impaired neurotransmitter release. BoNT types and certain subtypes differ in receptor recognition and/or intracellular substrate cleavage, leading to variable biological activity. BoNTs are associated with non-toxic proteins (ANTPs), including hemagglutinins (HAs) or OrfX proteins, to form complexes of various sizes conferring higher resistance to acidic pH and protease degradation. BoNTs are classically produced by anaerobic spore-forming bacteria from the genus Clostridium. The first toxigenic strain, which was isolated from an outbreak of human botulism in Ellezelles by van Ermengen in 1895, was named Bacillus botulinus. The term Clostridium was then used to designate these anaer-
obic spore-forming bacteria by the Committee on Classification of the Society of American Bacteriologists as opposed to Bacillus species, which only includes aerobic or facultative anaerobic microorganisms. It was then recognized that the clostridia involved in human and animal botulism synthesize immunologically distinct paralytic neurotoxins and are phenotypically and genetically diverse. Given the importance of the medical aspect of the clostridia responsible for botulism, the taxonomic position of Clostridium botulinum was originally based on only one phenotype, the production of a flaccid paralytic neurotoxin. However, physiological and genetic differences between C. botulinum strains led to the division of this species into four groups: (1) group I: C. botulinum A and proteolytic strains of C. botulinum B and F; (2) group II: C. botulinum E and glucidolytic strains of C. botulinum B and F; (3) group III: C. botulinum C and D; and (4) group IV: C. botulinum G, which was assigned to a new species C. argentinense. The taxonomic position of C. botulinum became more ambiguous since BoNTs can be produced by Clostridium strains clearly distinct from already defined C. botulinum, such as Clostridium butyricum and Clostridium baratii. The neurotoxigenic C. butyricum and C. baratii strains are phenotypically and genetically related to the typical strains of these species and are assigned to groups V and VI, respectively. More recently, novel BoNT types called BoNT/H (or FA or HA) and BoNT/X produced by C. botulinum strains have been reported (Barash and Arnon, 2014; Zhang et al., 2017). Increasing sequencing of individual bont genes and full genomes of C. botulinum strains support the distinction of the different BoNT
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Cell Host & Microbe
Previews types and their subdivision into more than 40 subtypes (Peck et al., 2017). Sequence variation between BoNT subtypes of a same type might impact on recognition by monoclonal antibodies for detection or therapeutic purpose and/or on biological activity. For example, BoNT/A2 induces a more rapid onset of local paralysis than BoNT/A1, A3, A4, and A5, and BoNT/F5 cleaves VAMP at a different site than the other BoNT/F subtypes. The localization of bont and antp genes on mobile DNA structures (plasmid, phage, transposon, or transposon-like DNA elements) supports horizontal neurotoxin gene transfer between Clostridium strains (Peck et al., 2017). Are bont genes restricted to Clostridium genus? In 2015, searching in genome databases allowed identification of a bont-related gene in non-clostridial bacterium, Weisella oryzae, which is a Gram-positive, non-spore-forming bacterium from fermented rice (Mansfield et al., 2015). The novel BoNT-like protein (BoNT/Wo) cleaves VAMP at a novel site and might be representative of a novel lineage of bacterial neurotoxins (Zornetta et al., 2016). In addition, a bont-related sequence gene has been found in a Gram-negative bacterium, Chryseobacterium piperi, from fresh water sediment, but the production of a biologically active neurotoxin has not yet been shown (Wentz et al., 2017). In this issue of Cell Host & Microbe, Zhang et al. (2018) show that a novel BoNT-related neurotoxin (BoNT/En) is produced by Gram-positive cocci from the Enterococcus genus. Zhang et al. (2018) found that an Enterococcus faecium strain isolated from cow contains a bont-related gene in an OrfX cluster that is located on a large plasmid. BoNT/En shares 29%–38.7% identity with the other BoNTs and shows a distinct enzymatic activity consisting of cleavage of both VAMP2 and SNAP-25 at novel cleavage sites. This novel BoNT also recognizes distinct receptor(s) since full-length BoNT/En is not toxic for mice in contrast to the other BoNTs. However, the enzymatic domain BoNT/En Lc linked to
BoNT/A Hc is highly paralytic in mice, supporting the neurotoxicity of this novel toxin in vivo when appropriate receptors are present. Thus, a bont-related gene encoding a functional neurotoxin is spread not only in clostridial strains, but also in other bacterial genus. Similar to the discovery of Zhang et al. (2018), a BoNT in Enterococcus called BoNT/J was also recently reported by another group (Brunt et al., 2018). What is the origin of such a gene encoding such a specific protein interacting with the neuroexocytosis machinery of eukaryotic organisms? Is it from clostridial or non-clostridial origin? Based on amino acid sequence and structure similarity, BoNTs, as well as tetanus neurotoxin (TeNT), probably originated from a common ancestor gene. Accordingly, it has been supposed that BoNT and TeNT could have arisen from viral protease (DasGupta, 2006). Interestingly, bonts have evolved in diverse types and subtypes and disseminated in clostridial and non-clostridial bacteria, whereas only one type of TeNT is known, which is produced by only one Clostridium species, C. tetani. In C. botulinum and other neurotoxigenic bacteria, including E. faecium, a conserved gene encoding a non-toxic and non-hemagglutinin (NTNH) protein that is structurally related to BoNT and is involved in BoNT stability, lies directly upstream of bont. Therefore, both bont and ntnh have likely evolved from the duplication of a common ancestor gene. In contrast, no ntnh-like gene has been found in C. tetani genome, indicating that duplication of the ancestor neurotoxin gene did not occur in this Clostridium species. This further suggests a clostridial origin of a neurotoxin gene ancestor, with subsequent duplication into ntnh and bont, diversification into types and subtypes in C. botulinum, and horizontal transfer in other clostridial and non-clostridial species, whereas C. tetani retains a conserved non-duplicated copy of the neurotoxin gene. The localization of BoNT/En on a likely conjugative plasmid supports the dissemination of bont-like gene in non-clostridial
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species (Zhang et al., 2018). However, the spreading of bont in non-clostridial strains seems very rare. Increasing whole-genome sequencing and investigation of bont genes should allow a better understanding of the neurotoxin genes in the bacterial world and their evolution as well as possibly to discover BoNT variants with potential novel therapeutic applications. REFERENCES Barash, J.R., and Arnon, S.S. (2014). A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins. J. Infect. Dis. 209, 183–191. Brunt, J., Carter, A.T., Stringer, S.C., and Peck, M.W. (2018). Identification of a novel botulinum neurotoxin gene cluster in Enterococcus. FEBS Lett. https://doi.org/10.1002/1873-3468.12969. DasGupta, B.R. (2006). Botulinum neurotoxins: perspective on their existence and as polyproteins harboring viral proteases. J. Gen. Appl. Microbiol. 52, 1–8. Jankovic, J. (2017). Botulinum toxin: state of the art. Mov. Disord. 32, 1131–1138. Mansfield, M.J., Adams, J.B., and Doxey, A.C. (2015). Botulinum neurotoxin homologs in nonClostridium species. FEBS Lett. 589, 342–348. Peck, M.W., Smith, T.J., Anniballi, F., Austin, J.W., Bano, L., Bradshaw, M., Cuervo, P., Cheng, L.W., Derman, Y., Dorner, B.G., et al. (2017). Historical perspectives and guidelines for botulinum neurotoxin subtype nomenclature. Toxins (Basel) 9, 38. Wentz, T.G., Muruvanda, T., Lomonaco, S., Thirunavukkarasu, N., Hoffmann, M., Allard, M.W., Hodge, D.R., Pillai, S.P., Hammack, T.S., Brown, E.W., and Sharma, S.K. (2017). Closed genome sequence of Chryseobacterium piperi strain CTMT/ATCC BAA-1782, a Gram-negative bacterium with clostridial neurotoxin-like coding sequences. Genome Announc. 5, e01296-17. Zhang, S., Masuyer, G., Zhang, J., Shen, Y., Lundin, D., Henriksson, L., Miyashita, S.I., Martı´nezCarranza, M., Dong, M., and Stenmark, P. (2017). Identification and characterization of a novel botulinum neurotoxin. Nat. Commun. 8, 14130. Zhang, S., Lebreton, F., Mansfield, M.J., Miyashita, S.I., Zhang, J., Schwartzman, J.A., Tao, L., Masuyer, G., Martinez-Carranza, M., Stenmark, P., et al. (2018). Emergence of a botulinum neurotoxin-like toxin in a commensal strain of Enterococcus faecium. Cell Host Microbe 23, this issue, 169–176. Zornetta, I., Azarnia Tehran, D., Arrigoni, G., Anniballi, F., Bano, L., Leka, O., Zanotti, G., Binz, T., and Montecucco, C. (2016). The first non-clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain. Sci. Rep. 6, 30257.
