Thuringiensin: a toxin from Bacillus thuringiensis  

Shana Leticia Felice Wiest , Harry Luiz Pilz Júnior , Lidia Mariana Fiuza
Laboratório de Microbiologia e Toxicologia, Universidade do Vale do Rio dos Sinos – UNISINOS, Av. Unisinos, 950, CEP 93001-970, São Leopoldo, RS, Brazil
Author    Correspondence author
Bt Research, 2015, Vol. 6, No. 4   doi: 10.5376/bt.2015.06.0004
Received: 23 Jun., 2015    Accepted: 28 Jul., 2015    Published: 14 Aug., 2015
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Wiest S.L.F., Pilz Júnior H.L. , and Fiuza L.M., 2015, Thuringiensin: a toxin from Bacillus thuringiensis, Bt Research, Vol.6, No.4 1-12 (doi: 10.5376/bt.2015.06.0004)

Abstract

Bacillus thuringiensis (Bt) is an entomopathogenic bacteria widely used in practice for biological control of insect pests, nematodes and disease vectors. Your toxicity is related to the ability to produce many virulence factors, including thuringiensins (β-exotoxins). Most toxins produce by this microorganism are highly specific, therefore the use of  it in biological control is considered environmentally safe and so this bacteria is extensively used in the production of biological insecticides and genetically modified plants. However, the thuringiensin is considered toxic to almost all life forms, including humans, due to its ability to inhibit the biosynthesis of RNA polymerase, an enzyme essential to the transfer of genetic information in almost all organisms. This way, the release of new strains of Bt with insecticidal properties for the biological control of pests must pass by verification of the absence of production of exotoxin, so that non-target organisms are not affected and the use of Bt in this field remains safe. Thus, this revision will discussed the knowledge about features, structure, genetic determinants, biosynthesis, mechanism of action, insecticide spectrum, security assessment and procedures for identification of thuringiensin in Bt strains.

Keywords
Biological control; environmental security; ?-exotoxins; thermostable toxin

Background
Bacillus thuringiensis Berliner (1911) (Bt) (Eubacteriales: Bacillaceae) are a ubiquitous soil bacteria and was first isolated in 1901, in Japan, by Shigetane Ishiwata, in sick larvae of Bombyx mori (Lepidoptera: Bombycidae) (silkworm), being initially named sotto disease. Seven years later, Iwabuchi (1908) called it as Bacillus sotto. In 1911, the same bacteria was found in the province of Thuringia, Germany, by bacteriologist Ernst Berliner from infected larvae of Ephestia kuehniella (Lepidoptera: Pyralidae) (moth flour-of-Mediterranean) (Bravo et al., 2012; Ramirez-Lepe and Montserrat 2012). To analyze it, Berliner reported the presence of parasporal protein crystals (Cry), but did not know how to describe the role of these proteins in this microorganism, which remained unknown until 1950 (Federici et al., 2010). Currently, it is known that Bt can establish lethal infections in susceptible organisms to break the midgut cells and replicate itself inside the hemolymph of living host (Raymond et al., 2010).
Because of their entomopathogenic properties, Bt is widely used in biological control of insect pests practices, nematodes and vectors of human diseases (Bravo et al., 2012). The first tests in field using this bacterium were carried out in 1929, by Husz, in corn fields, for the control of Ostrinia nubilalis larvae (Lepidoptera: Crambidae) (European corn worm). The positive results of the experiments caught the attention of agribusiness industries in this entomopathogenic organism (Kaya and Vega, 2012). The first commercial formulation of Bt, the insecticide spray Sporeine, was developed in France in 1938, but had low selectivity and action limited to few species. The large-scale production was started only in the 50s, reaching countries such as Russia, Czechoslovakia, France, Germany and the United States. However, few strains of Bt were used for the production of spray insecticides to date, representing about 2% of the marketable insecticides (Pardo-López et al., 2013).

On the other hand, with the development of molecular techniques and biotechnological advances, it has become possible the genetic manipulation of plants, through the insertion of small sequences of genes from B. thuringiensis expressing toxic proteins of the bacterium directly into the genotype of the plant (Pinto-Zevallos e Zarbin, 2013). In the early 80s, Schnepf and Whiteley (1981) conducted the first cloning and sequencing of the first genes encoding these proteins, obtained through an isolated from Bt subsp. kurstaki strain and expressed it initially in tobacco and tomato plants, which were the first transgenic-Bt plants to be marketed (Ali et al., 2010; Rodríguez et al., 2012). Thus, it was possible the introduction of Bt genes in other plants to introduce resistance to insect pests, such as corn and cotton cultivars which are the two major Bt crops grown worldwide (Ali et al., 2010).

The main toxins produced by Bt presenting entomopathogenic activity are the δ endotoxins (Cry and Cyt) and parasporins, which are intracellular proteins; besides the α exotoxins; thuringiensins (β-exotoxins); toxins VIPs (vegetative insecticidal proteins); S-layer proteins (SLP) and exoenzymes (lipases, proteases, chitinases and phospholipase C), which are extracellular macromolecules enabling virulence (Arora et al. 2013; Bravo et al. 2011; Vu et al., 2012). As biolarvicides, Bt proved to be innocuous in tests that evaluated the potential toxicity of the most of their toxins in mammalian cells, and organisms non-target (Thomas and Ellar, 1983). All of these toxins have been studied for its potential use in biological control practices. However, not all can be used in pest control, such as the thuringiensin, which has a broad spectrum of biological toxicity to a variety of non-target species, including mammals. Therefore, these present review discuss current knowledge about characteristics, types, genetic determinants, biosynthesis, mode of action, insecticide spectrum, safety assessment and procedures to identification of thuringiensins in Bt strains.

1 Features of Thuringiensins
Thuringiensins are secondary metabolites, nonproteic and soluble in water. It is also a heat-stable exotoxin that maintains their bioactivity to 121? for 15 min (Farkas et al., 1969). It is known that are more stable at pH 7.0, but their stability decreases with increasing temperature (Zhou et al., 2013). As in other extracellular insecticides toxins, their production occurs during the vegetative growth phase of some strains of Bt, when are secreted in the culture medium where the bacteria is inoculated (Liu et al., 2010; Obeidat et al., 2012). However, according to (Argôlo-Filho et al., 2014), the secretion of this secondary metabolite varies temporally, thus the knowledge of the temporal pattern of secretion or activity in the culture medium is necessary to avoid the lack of identification of this undesirable exotoxin. The different toxicity scale of thuringiensin, highly dependents of the time of cultivation of the producing strain, may also be explained by different volumes of culture, and the variable conditions of the culture medium (aeration level; pH value) (Argôlo-Filho et al., 2014). (Jing-Wen et al., 2007) reported that the pH and the concentration of glucose had an important effect on the synthesis and efficiency of thuringiensin. Physiological differences between strains can also result in a variation of the secretion of toxins, even when it is inoculated the same quantities of cells, generating toxicity profiles temporally distinct for each strain, and, consequently, a potential wrong classification of producer isolates (Argôlo-Filho et al., 2014).

The exotoxin thuringiensin was discovered by McConnell and Richards (1959), which describe it as a substance thermostable toxic to insects. Heimpel (1967) proposed the name β exotoxin to designate it, but over time, this term was considered inappropriate, due to the structure of this toxin. Instead, several authors suggested the thuringiensin synonym that is currently used (Kim and Huang, 1970; Pais and De Barjac, 1974; Farkas et al., 1977).

The chemical formula of the thuringiensin is C22H32N5O19P (De Rijk et al., 2013). Initially, (Farkas et al., 1969) reported that the structural formula of thuringiensin consisted of adenosine, glucose, a phosphoric acid and a gluconic diacid. Šebesta and Horska (1970) suggested that exotoxin is composed of adenine, aleric acid phosphorylated and a sugar moiety formed by D-ribose and D-glucose linked by an ether unusual. Currently, it is known that the thuringiensin has a unique structure and, as a polymer of monosaccharides, has asymmetric carbon atoms (Liu et al., 2010) (Figure 1).

 
Figure 1 Structure of thuringiensin I (Belder e Elderson. 2013) 


Many Bt strains belonging to different serotypes excrete the toxin (Jing-Wen et al., 2007), as well as some strains of B. subtilis, and B. megaterium (Pinto et al., 2010). Some studies reported that strains of B. cereus also are able of producing thuringiensins (Carlberg 1986; Krieg and Lysenko 1979; McConnell and Richards 1959; Ohba et al., 1981). Perchat et al., (2005), for example, performed a screening of 575 strains of B. cereus and found 270 strains producers of thuringiensins of type II.

2 Types of Thuringiensins
Levinson et al., (1990) described two types of thuringiensins from assays of high-performance liquid chromatography (HPLC). Thuringiensins of the type I have low molecular weight, approximately 701Da, and are composed of adenosine, glucose, a phosphate group and gluconic diacid (Liu et al. 2010; Mac Innes and Bouwer 2009). For many years it was believed that thuringiensin I was a phosphorylated molecule analogous to the adenine nucleotide with great structural similarity to this nucleotide (Šebesta and Horska 1970; Šebesta and Sternbach 1970; Šebesta et al., 1981). However, recently, Liu et al., (2010) proposed that, in fact, it is an oligosaccharide from adenine nucleoside.

Because of the similarity to adenine, the toxicity of thuringiensin I was explained by the inhibition of RNA polymerase biosynthesis, one of the key enzymes in the transfer of genetic information. That's because this exotoxin acts essentially in the step of the polymerization of the polymerase reaction, competing for binding sites with ATP (Devidas and Rehberger 1992; Perchat et al., 2005). More specifically, thuringiensins I bind reversibly (without being incorporated into the polymer) to a portion of adenosine in the specific site of ATP (Šebesta and Sternbach 1970). The depression of RNA polymerase biosynthesis was seen in assays with rats (Šebesta and Horska 1969), and as it is a fundamental process for all types of life, thuringiensins I are toxic to almost all living organisms (Belder and Elderson 2013).

Levinson et al., (1990), analyzing strains of Bt subsp. thuringiensis, Bt subsp. tolworthi and Bt subsp. darmstadiensis, reported that the gene that encodes the thuringiensin I production is supported by plasmids. Ozawa and Iwahana (1986) provided evidences that the production of exotoxin is associated with a plasmid of 62 Mdal in a strain of Bt subsp. darmstadiensis, but that plasmids producers thuringiensins I are not ubiquitous in Bt strains. Initially, it was believed that an ABC transporter could be related to the secretion and production of this exotoxin (Espinasse et al., 2002a). The researches about thuringiensins are focused on tests to measure their insecticidal activity and in strategies for its detection and purification, because studies on genetic determinants involved in its biosynthesis are still scarce (Liu et al., 2014). Recently, it was discovered that the Thu3 gene, which is homologous to ABC transporter is involved in the secretion thuringiensin I. According to Liu et al., (2010) synthesizers gene of this exotoxin are encoded by circular endogenous plasmids of 110 kb that harboring thu cluster besides synthesizers genes of the Cry1Ba proteins (Iatsenko et al., 2014). More specifically, in the thuringiensin biosynthesis, genes thuA, thuC and thuD encode proteins responsible by the synthesis of the key precursor of the toxin, a gluconic diacid (precursor A) from glucose-6-phosphate. The thuF and thu1 genes encode proteins involved in the assemblage of thuringiensin I. The thuE gene encodes the enzyme responsible for the synthesis and phosphorrylation of the toxin and the thu3 gene encodes a protein that acts in the release of thuringiensin I mature, which may be secreted by the type IV-like secretion system (T4SS)  towards the cell (Liu et al., 2010) (Liu et al., 2010).

Some studies report that the thuringiensin I production is related to the presence of plasmids that also harbor cry and vip1/vip2 genes (Espinasse et al. 2002b; Cstagnola and Stock 2014; Iatsenko et al., 2014; Levinson et al., 1990). (Espinasse et al., 2002a) and (Perani et al., 1998), for example, report that the production of high levels of thuringiensin I is linked to the presence of plasmids carrying the gene encoding the crystal protein Cry1b. However, Bt strains that do not produce crystals may also produce thuringiensins I, such as mutant strain of B. thuringiensis 407-1 (Cry-), which still synthesizes a pigment of soluble melanin also secreted in the culture supernatant. Therefore, the production of thuringiensin I can occur even in a lineage that lost the plasmids containing the genes cry (Espinasse et al., 2002b). It is believed that these endogenous plasmids encoders thuringiensins I were acquired by horizontal gene transfer, in a co-evolution with insects through a host-parasite relationship. Representing a unique genetic resource and as part of adaptive genetic pool can play an important role in the biology and evolution of Bt where cells are hosted (Schnepf et al., 1998).

(Levinson e

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