Neurotoxins from Clostridium botulinum (serotype A) isolated from the soil of Mendoza (Argentina) differ from the A-Hall archetype and from that causing infant botulism
Introduction
Clostridium botulinum (Cb) is a spore-forming Gram positive bacillus that produces botulinum neurotoxins (BoNT). BoNT are responsible for the fatal neuroparalytic disease botulism. BoNT enter nerve terminals and cause a prolonged neurotransmitter exocytosis blockade, resulting in an impairment of muscle contraction and autonomic nerve functions (reviewed by Zhang et al., 2010). To date, two forms of the disease have been described: the one caused by the preformed toxin, including botulism caused by contaminated food intake, and the toxo-infection which is caused by bacterial colonization of the intestine and the subsequent in-situ release of BoNT. The latter category includes the infant botulism (IB), which is one of the most frequent forms of botulism worldwide affecting infants under one year of age (Cox and Hinkle, 2002, Koepke et al., 2007, Fenicia and Anniballi, 2009). Spores of BoNT-producing clostridia are present in the environment and can be found in the dust both domiciliary and peridomiciliary, where the soil appears to be the most important source of contamination (Thompson et al., 1980).
Seven serotypes (A-G) of BoNT have been identified based upon their antigenicity (Arnon et al., 1979). Each serotype is produced by a different strain of Clostridium botulinum (Cb), with all exhibiting a high amino-acid sequence homology (Hill et al., 2007; reviewed by Peng Chen et al., 2012). Four serotypes (A, B, E and rarely F) are known to cause human botulism (reviewed by Hambleton, 1992, Montal, 2010, Peng Chen et al., 2012). In turn, several subtypes have been identified from these serotypes (Smith et al., 2005, Arndt et al., 2006, Carter et al., 2009, Umeda et al., 2009, Jacobson et al., 2011, Peng Chen et al., 2012). Each BoNT is synthesized as a harmless single polypeptide chain with a molecular mass of ∼150 kDa (protoxin). The inactive precursor protein is cleaved by proteases into two active domains, a 50-kDa light chain (LC) and a 100 kDa heavy chain (HC) linked by an interchain disulfide bond (holotoxin). After reduction of the disulfide bond, the fully active toxin is generated. However, to reach their targets (peripheral nerves), toxins need to cross the epithelial barrier of the digestive tract. To this end, BoNTs associate with non-toxic, non-hemagglutinin (NTNH) and some also with hemagglutinin components (NTH). This association gives rise to three complex forms; a protein of 300 kDa (12S) associated with NTNH (toxin M), a protein of 600 kDa (16S) associated with both NTNH and NTH components (toxin L), and a 19S complex, which is thought to be a 16S dimer (toxin LL) (reviewed by Fujinaga, 2010). The Cb serotype A, (subtype A1 and A5) produces the three types of complexes (Carter and Peck, 2015), while serotypes B, C and D only produce the 12S and 16S complexes. In turn, the subtype A2-A4, and the serotypes E and F, only produce the 12S complex (Sakaguchi, 1982, Oguma et al., 1999, Poulain et al., 2008, Carter and Peck, 2015).
It is believed that the component NTNH confers proteolytic resistance to BoNTs in the gastrointestinal tract, whereas NTH may play a role in the toxin internalization by the intestine epithelial cells (Fujinaga et al., 1997).
Although the lethal doses of BoNTs in humans are not known, they are assumed to be very similar to those of mice, except for BoNT/D, which is not harmless to humans (Coffield et al., 1997). Arnon et al. (2001) have estimated a LD50 of 1 ng/kg in humans.
BoNTs cause flaccid paralysis through an acetylcholine release blockade at the neuromuscular junction in peripheral α-motor neurons. This blockade is accomplished through the cleavage of SNARE proteins (reviewed by Anhert-Hilga et al., 2013) by the LC zinc metalloprotease domain. Thus, serotypes A, C, and E cleave SNAP25, while serotypes B, D, F, and G cleave VAMP-2, and serotype C also cleaves syntaxin 1a (Schiavo et al., 2000, Zhang et al., 2010).
Epidemiological and molecular studies have correlated the prevalence of clostridia producing different botulinum neurotoxin serotypes with geographical regions, and with the presence of disease. Thus, in Argentina the serotype A is prevalent and mostly linked to IB (Lúquez et al., 2005), being the principal route of entry through ingestion of spores from the soil.
In order to identify the soil (a Cb reservoir) as the main infection source for IB in the province of Mendoza (Argentina), we characterized and compared Cb strains and their respective neurotoxins in bacterial isolates obtained from the soil and those obtained from the fecal samples of IB patients.
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Obtention of native strains of C. botulinum
Ten native strains of Cb were isolated, five from fecal samples obtained from infants with botulism (IBCb) and five from soil samples (SCb) of different areas in the province of Mendoza (Argentina) (Bianco et al., 2008, Bianco et al., 2009). Strain A-Hall was used for comparison. These strains were maintained lyophilized until used. Strains were resuspended in chopped-meat medium (Giménez and Ciccarelli, 1970) under anaerobic conditions. After 48 h incubation at 35 °C, the purity of strains was
Morphological characterization of the isolated colonies
The SCb strain (Fig. 1 A), but not the IBCb strain (not shown), displayed a swarming growth pattern as a thin film on 1.5% agar. When grown on 4.0% agar, SCb strains evinced 2–2.2 mm diameter colonies, with a predominantly irregular shape with high, refringent borders, and with an opaque, and depressed center (Fig. 1B). The IBCb strain formed smaller colonies (1.2–1.5 mm diameter) with refringent, mottled margins, and a mottled and an embossed center (Fig. 1C), similar to the A-Hall archetype (
Discussion
Infant botulism (IB) has been documented in 26 countries in four continents, with Argentina reporting the second highest number of cases (Fenicia and Anniballi, 2009). Many studies suggest that the main source of contamination are the Cb spores resident in the soil, although contaminated honey has been associated with a small number of cases in different countries (Fenicia and Anniballi, 2009). In addition, the presence of BoNT-producing clostridia spores has also been detected in samples of
Acknowledgments
Dr. Miguel Sosa is a Career Researcher of CONICET. We thank, Mr. T. Sartor for his valuable technical assistance. This study was supported by the Grant J004 from SECTyP (National University of Cuyo, Mendoza, Argentina).
References (46)
- et al.
Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins
Biochem. Biophys. Res. Commun.
(2005) - et al.
A structural perspective of the sequence variability within Botulinum Neurotoxin subtypes A1-A4
J. Mol. Biol.
(2006) - et al.
Honey and other environmental risk factors for infant botulism Infectious Disease
J. Pediatr.
(1979) - et al.
Presence of Clostridium botulinum spores in Matricaria chamomilla (chamomile) and its relationship with infant botulism
Int. J. Food Microbiol.
(2008) - et al.
Genomes, neurotoxins and biology of Clostridium botulinum group I and group ii
Res. Microbiol.
(2015) - et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951) - et al.
Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1
FEBS Lett.
(2011) - et al.
Developmental differences between cation-independent and cation-dependent mannose-6-phosphate receptors in rat brain at perinatal stages
Dev. Brain Res.
(2005) - et al.
Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds
FEBS Lett.
(1993) Clostridium botulinum toxins
Pharmacol. Ther.
(1982)
Cleavage ofmembers of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin
J. Biol. Chem.
Synaptic vesicle protein: targets and routes for botulinum neurotoxin Curr
Top. Microbiol. Immunol.
Botulinum toxin as a biological weapon: medical and public health management
J. Am. Med. Assoc.
Linden flower (Tilia spp.) as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism
Rev. Argent. Microbiol.
Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum
BMC Genomics
Genus Clostridium, praemozski 1880, 23AL
In vitro characterization of botulinum toxin types A, C and D action on human tissues: combined electrophysiologic, pharmacologic and molecular biologic approaches
J. Pharmacol. Exp. Ther.
Infant botulism
Am. Fam. Physician
Selective medium for isolation of Clostridium botulinum from human feces
J. Clin. Microbiol.
Infant botulism
Ann. Ist. Super. Sanita
The haemagglutinin of Clostridium botulinum type C progenitor toxin play an essential role in binding of toxin epithelial cell of Guinea pig small intestine, leading to the efficient absorption of the toxin
Microbiology
2010 Interaction of botulinum toxin with the epithelial barrier
J. Biomed. Biotechnol.
Distribución de Clostridium botulinum en Mendoza, Argentina
Bol. Sanit. Panama
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They contributed equally to this work.