Elsevier

Toxicon

Volume 121, October 2016, Pages 30-35
Toxicon

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

https://doi.org/10.1016/j.toxicon.2016.08.010Get rights and content

Highlights

  • Native strains of Clostridium botulinum were isolated from soil or infant botulism feces and studied comparatively.

  • The neurotoxins purified from these strains are similar at molecular level but different than that of A-Hall strain.

  • The neurotoxin from soil Clostridium botulinum showed to be more potent than that from infant botulism.

  • The neurotoxins from both strains can cleave SNARE proteins differentially.

Abstract

The type A of neurotoxin produced by Clostridium botulinum is the prevalent serotype in strains of Mendoza. The soil is the main reservoir for C.botulinum and is possibly one of the infection sources in infant botulism. In this study, we characterized and compared autochthonous C. botulinum strains and their neurotoxins. Bacterial samples were obtained from the soil and from fecal samples collected from children with infant botulism. We first observed differences in the appearance of the colonies between strains from each source and with the A Hall control strain. In addition, purified neurotoxins of both strains were found to be enriched in a band of 300 kDa, whereas the A-Hall strain was mainly made up of a band of ∼600 kDa. This finding is in line with the lack of hemagglutinating activity of the neurotoxins under study. Moreover, the proteolytic activity of C. botulinum neurotoxins was evaluated against SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins from rat brain. It was observed that both, SNAP 25 (synaptosomal-associated protein 25) and VAMP 2 (vesicle-associated membrane protein) were cleaved by the neurotoxins isolated from the soil strains, whereas the neurotoxins from infant botulism strains only induced a partial cleavage of VAMP 2. On the other hand, the neurotoxin from the A-Hall strain was able to cleave both proteins, though at a lesser extent. Our data indicate that the C.botulinum strain isolated from the soil, and its BoNT, exhibit different properties compared to the strain obtained from infant botulism patients, and from the A-Hall archetype.

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.

Section snippets

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).

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