Bacillus thuringiensis: mechanism of action

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http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.960793

REVIEW ARTICLE

Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Mrs Claire Summerfield on 10/14/14 For personal use only.

Andre´ Luiz de Almeida Melo, Vanete Thomaz Soccol, and Carlos Ricardo Soccol Programa de Po´s Graduaçaõ em Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Parana´, Curitiba, Parana´, Brazil

Abstract

Keywords

Since the first report by Ishiwata in 1902 of a Bombyx mori infection, followed by the description by Berliner, Bacillus thuringiensis (Bt) has become the main microorganism used in biological control. The application of Bt to combat invertebrates of human interest gained momentum with the growing demand for food free of chemical pesticides and with the implementation of agriculture methods that were less damaging to the environment. However, the mechanisms of action of these products have not been fully elucidated. There are two proposed models: the first is that Bt causes an osmotic imbalance in response to the formation of pores in a cell membrane, and the second is that it causes an opening of ion channels that activate the process of cell death. There are various ways in which Bt resistance can develop: changes in the receptors that do not recognize the Cry toxin, the synthesis of membrane transporters that eliminate the peptides from the cytosol and the development of regulatory mechanisms that disrupt the production of toxin receptors. Besides the potential for formulation of biopesticides and the use in developing genetically modified cultivars, recent studies with Bt have discussed promising applications in other branches of science. Chitinase, an enzyme that degrades chitin, increases the efficiency of Bt insecticides, and there has been of increasing interest in the industry, given that its substrate is extremely abundant in nature. Another promising field is the potential for Bt proteins to act against cancer cells. Parasporins, toxins of Bt that do not have an entomopathogenic effect, have a cytotoxic effect on the cells changed by some cancers. This demonstrates the potential of the microorganism and new opportunities opening for future applications.

Apoptosis, cancer, chitinase, cry toxin, pore-forming

Introduction Bacillus thuringiensis (Bt) was first identified in the beginning of the twentieth century in 1902 by Ishiwata, who reported the microorganism infecting Bombyx mori and causing damage in the silk industry of Japan (Beegle & Yamamoto, 1992). At that time, the author named it Bacillus sotto, which means soft and flabby, in reference to the appearance of the infected larvae. Subsequently, in the city of Thuringia (Germany), Berliner isolated a Gram-positive bacterium in the moth Ephestia kuehniella larvae, and, ignoring the nomenclature of Ishiwata, named it Bacillus thuringiensis. This name has persisted until now. Although Berliner proved the toxic effects of repeated ingestion, leading to the death of insects, the author did not consider the applicability of Bt to the control of moth larvae, since at that time little was known about the characteristics or potential of the Bt bacteria (Coˆte´, 2007). The microorganism was again isolated by Mattes in 1927 and was used in the

Address for correspondence: Carlos Ricardo Soccol, Programa de Po´s Graduaçaõ em Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Parana´, Curitiba, Parana´, Brazil. E-mail: [email protected]

History Received 9 April 2014 Revised 22 July 2014 Accepted 24 July 2014 Published online 26 September 2014

following years for the biological control of Ostrinia nubilalis (Lepidoptera: Crambidae) (Beegle & Yamamoto, 1992). The first formulation of Bt biopesticide emerged in the following decade, and the product Sporeine was applied to control the various Lepidoptera species that adversely affected crops in France (Milner, 1994). Since then, the spectrum of susceptible organisms has expanded and now also includes the orders of Culicidae, Coleoptera, Simuliidae, Hymenoptera, Homoptera, Mallophaga and others (Sanchis, 2012). Subsequently, with the advent of molecular biology, crystallized genes of Bt toxins were inserted into cultivars, making these genetically modified plants resistant to various pests (Roh et al., 2007). Despite the potential of Bt to be exploited for a long time as a pest control, for the production of genetically modified plants, and to control the vector of human diseases, research on this microorganism is still incomplete. Every year, new approaches are found for the study of bacteria, and these pave the way for new applications (Bravo et al., 2007; Dash et al., 2013; Ernandes et al., 2012; Kroeger et al., 2013; Tan et al., 2010). This review focuses on recent discoveries involving the mechanism of action of Bt, the study of toxin resistance, the resistance management and an overview of the current studies on this bacteria and its applications, such as the production of chitinases and its use as a toxin for cancer cells.

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Mechanism of action by B. thuringiensis Although Bt-derived products have been successfully used for a long time to control a large number of invertebrates, the mechanisms that induce cytotoxicity have not been fully elucidated. Although new bacterial strains are isolated daily and in vivo tests attest to the susceptibility of different species, studies that unveil the events that result in cell death are not progressing at the same speed. Currently, it is known from surveys that a specific strain of Bt can be used to combat a specific species (which is usually a Lepidoptera species of interest in agriculture; Bravo et al., 2011; Cooper et al., 1998; Jurat-Fuentes & Adang, 2006; Schwartz et al., 1997). In these studies, it can be noted that several mechanisms of toxicity have been reported, indicating that there is no single standard model. Furthermore, these complex processes may occur simultaneously, and they show that there are still gaps in our knowledge of the d-endotoxin receptors in the plasma membrane and the sequence of reactions that culminate in the death of an organism. We detail below two mechanisms among many others where the death occurs. Sequential binding model The current model for the cytotoxic action of B. thuringiensis is that pores form in the plasmatic membrane, which results in sequential binding and damages the osmotic balance (Figure 1). This is known as the classic mechanism, and in recent years, it has been extensively detailed by leading researchers (Bravo et al., 2007; Pardo-Lo´pez et al., 2009; Soberoń et al., 2010). This has been detailed in studies with Cry1Ab in Manduca sexta: the d-endotoxin is ingested, the digestive protease is activated, and then the Cry toxin comes into contact with the N-aminopeptidase and cadherin receptors on the surface membrane (Dorsch et al., 2002; Hua et al., 2004; Jimeńez-Jua´rez et al., 2008; Figure 1A). Specific molecular affinities between the toxins and certain receptors result in proteolytic cleavage in the Cry molecule. This causes structural changes in the chain and forms oligomers that function as ‘‘pre-pores’’ (Figure 1B and C). There is also a third connection to the membrane surface: the N-aminopeptidase receptor has a molecular affinity and acts to anchor the pre-pore in the lipid bilayer (Figure 1D). Thus, the change in the pore formation affects the integrity of the

Figure 1. Mechanism of action by the Bt Cry toxin, according to the sequential binding model. (A) The Cry toxin (Tx) binds the cadherin receptors (Cd) in the plasma membrane. (B) The Cry toxin connects the N-aminopeptidases receptors (N-a). (C) Proteolytic is cleaved by the oligomerization of the protein (Ol). (D) Pre-pore formation and membrane anchoring. (E) Pores (P) that affect the integrity of the membrane.

Crit Rev Biotechnol, Early Online: 1–10

membrane (Figure 1E), and there is a consequent loss of function. The osmotic imbalance then leads to cell death. The model of sequential binding considers the links between toxins and receptors and views the toxin as molecularly altering the protein and triggering pores in the body that is susceptible to cytotoxicity. The model is based on cases of resistance in which structural changes in the receptor impedes cytotoxicity. However, it does not explain some types of resistance, such as when tolerance occurs without any change in the structure of the receptor (Vachon et al., 2012). Similarly, the silencing of RNA cadherin and N-aminopeptidases triggers Cry resistance, confirming the importance of these components in the biological activity of Bt (Schwartz et al., 1997). However, the expression of only one of these heterologous receptors (cadherin or N-aminopeptidase) is enough to render the cell again sensitive to various types of Cry1A toxin (Fabrick et al., 2009). This shows that there may be other routes to resistance and toxicity. Another critical point in the theory is the formation of prepores, which occurs when there is a cleavage of an a-helix protein. In experiments, it was observed that the rate of pore formation was not influenced by the activity of proteases; i.e. the stimulations of toxin cleavage does not directly affect the formation of pores, as would be expected to occur in the midgut of larvae (Baxter et al., 2005; Go´mez et al., 2002). Moreover, even if the cleavage sites are not involved in removing the a-helix, which would inhibit the formation of pre-pores, it appears that the process still occurs, but at a slower rate. One study showed that excessive Cry1Ab proteolysis hindered the formation of pores (Lebel et al., 2009), and this demonstrated that it is necessary for the sequential occurrence of the reactions in the binding model and the formation of pores in the cell membrane. Signaling pathway model The second proposed mechanism has some similarities with the previous model; however, it assigns other causes for cell death. According to this theory, the Cry proteins affect the cell in two ways: first, in a lytic manner, forming pores in the membrane, as in the sequential binding model; second, producing successive reactions that alter cellular metabolism (Schwartz & Laprade, 2000; Zhang et al., 2005; Figure 2).

Figure 2. Mechanism of action by Bt toxin, according to the signaling pathway model: (A) The Cry toxin (Tx) binding to cadherin receptors (Cd) in the plasma membrane; (B) Stimulating apoptosis (Ap) with activation of ions channels (ci) in the plasma membrane; (C) Movement of magnesium ions, culminating in cell death.


DOI: 10.3109/07388551.2014.960793

While the formation of pores determines the final result of the toxin, in this model, the importance of incorporating a prepore into the membrane is minimized. It is not considered sufficient to cause cell death, and it is not always coupled with the membrane lipids (Zhuang et al., 2002). Furthermore, in some cases, bacterial toxins simply do not bind the membrane to form pores, although cytotoxicity occurs in the same way. According to this hypothesis, the Cry toxin receptors bind to cadherin and N-aminopeptidases (Figure 2A), and, by an unknown processes, transmit stimuli that result in the activation of Mg2+ channels in the plasma membrane (Figure 2B). The opening of these channels causes abnormal movement of ions in the cytosol (Figure 2C). The effect of this is much larger due to the destruction of the plasma membrane, and bacterial toxins activate pre-existing cellular processes during the apoptose (Zhang et al., 2005). According to this model, a lytic form can occur when the oligomers are inserted into the lipid bilayer, but this will not be cytotoxic. Analysis of the signaling pathway model shows major gaps in our understanding of the action mechanism. By simplifying the long process of toxin activation and its connection to the receiver, the model neglects the interactions of the Cry protein, d-endotoxin and membrane receptor, which have been extensively studied (Vachon et al., 2012). Both in vitro and in vivo studies have successfully caused membranes to be permeable, demonstrating the success of forming pores in an intact bilayer lipids membrane, yet the signaling pathway considers this from another perspective. The connection between toxins and receptors would not be important in the formation of pores, except for the involvement of ion channels in the activation of cell necrosis. Previous studies have noted Cry activation of the Ca2+ channel protein, demonstrating permeabilization and intracellular signaling effects (Pe´rez et al., 2007; Rausell et al., 2004; Zhuang et al., 2002). However, the hypothesis that these ions are involved in the toxicity of Bt has not been sufficiently explored in connection with this model. Although the models that attempt to explain the mechanism of the toxicity of Cry serve as a basis for practical work, there is a need for further experimental verification. The signaling pathway model ignores an extensive literature on the receptors and bonds that occur during the processing of d-endotoxin, and this minimizes the importance of the cascade of events that cause the toxicity (Zhang et al., 2005). The model provides a standard sequence of processes during which the toxin binds to the receptors. However, it still does not sufficiently explain the mechanism of action in view of the wide variation in metabolic routes that may culminate in the resistance or death of a cell (Vachon et al., 2012). The studies seem to indicate that the Cry protein can cause both the formation of pores on membranes and the activation of ion channels.

Resistance to Bt toxins Resistance in Lepidoptera The first reports of resistance to a Bt insecticide occurred in the early 1990s. In this case, Plutella xylostella caterpillars (Lepidoptera: Plutellidae) were resistant to Dipel 2X (Abbott Laboratories North Chicago, OL), one of the first

Bacillus thuringiensis

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commercial formulation of Bt produced by the HD-012 strain. Later, this response was characterized as a reaction to Mode 1, which encompasses a particular pattern of resistance (Hernańdez-Rodrı´gues et al., 2012). Initially, in mode 1, there is a high resistance to at least one subtype of Cry1A protein (Cry1Aa, Cry1Ab and Cry1Ac) and low resistance to Cry1C. This selected Lepidoptera-resistant characteristic is defined by the inheritance of an autosomal recessive gene. In addition to this, there was also an observation of crossresistance, in which the tolerance to a particular. Cry toxin allows the development of resistance to another variant. For example, when Cry1A resistance results in resistance to Cry1F (Tan et al., 2013). In the following years, reports of resistance involving Bt insecticides and genetically modified cultivars with variants of the Cry toxin began to emerge in various parts of the globe. By tests in maize in Central American countries such as Guatemala, Honduras and Costa Rica, Perez & Shelton (1997) confirmed resistance of Plutella xylostella (Lepidoptera: Plutellidae) to a Bt formulation. Farino´s et al. (2004) observed the development of resistance in Sesamia nonagroides (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Lepidoptera: Pyralidae) in fields of GM maize in Spain. Kranthi et al. (2006), studying transgenic cotton crops in India, found that the resistance of Helicoverpa armigera (Lepidoptera: Noctuidae) was a semi-monogenic autosomal and dominant inheritance. In a study of this pest in crops of GM cotton in Australia, Mahon et al. (2007) reported the presence of two autosomal genes that confer resistance. Kruger et al. (2011) also observed resistance in Busseola fusca (Lepidoptera: Noctuidae) on GM corn crops with Cry2Ab in South Africa. The study of the mechanisms that determine resistance of insects to the toxin Cry gained momentum with the implementation of genetically modified cultivars, which is when it became vital to understand it in order to protect crops and ensure high productivity (Tabashnik et al., 2008). Thus, since 2000, there has been an effort to understand at a molecular level the resistance to Bt, and there have been several studies published (Morin et al., 2003). Among the various forms of resistance, one can be highlighted: a three-way combination of a failure to recognize cadherins, failure to synthesize membrane transporters and failure of the transregulatory mechanism (Table 1). Modification to the cadherin receptor In an early molecular-level study of the Bt toxin receptor, Morin et al. (2003) verified the existence of three alleles that encode the cadherin-related receptors that are resistant to Cry1Ac. In the resistant populations of Pectinophora gossypiella (Lepidoptera: Gelechiidae) that affect cotton crops, the authors reported the presence of t2 mutated alleles that result in caterpillars resistant to the Cry toxin. However, one or two normal alleles result in susceptible caterpillars, which indicate that this is recessive. The study revealed that the result of the three alleles was a cadherin from which eight amino acids were deleted in the quaternary structure of the receiver. This occurred precisely at the binding sites for Cry1Ac and so prevented the recognition and docking of the

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Table 1. Resistance strategies of Lepidoptera to tolerate Cry toxins from biological insecticides and Bt crops. Forms of resistance Modification to the cadherin receptor type Membrane transporters


Transregulatory mechanism

Mode of action

Cause

Reference

Amendment in the cadherins results in no activation by the toxin Cry Structure present in the plasma membrane performs active transport of the toxin out of the cytosol Alteration in the expression of aminopeptidases APN1 APN6 inhibits Cry1Ac

Mutation in the cadherin gene generates change in binding site

Morin et al. (2003) Zhang et al. (2005) Chen et al. (2009) Gahan et al. (2010) Baxter et al. (2011) Hernandez-Rodriguez et al. (2012)

toxin, which thus prevented the whole cascade of events that would result in cell death. The cadherin receiver also occupies a central position in the toxicity of Cry1Ac, according to Zhang et al. (2005). In a study of the Bt toxin in Manduca sexta (Lepidoptera Sphingidae), the authors reported that the activity of the toxin depends on the presence of the cadherin Bt-R1. If the receptor on the membrane is removed or altered, the surface is rendered resistant to Cry1Ac in the same way that the addition of BT-R1 would restore susceptibility. Chen et al. (2009) also determined that a cadherin receptor was responsible for the cytotoxic action of the Cry11Aa proteins produced by B. thuringiensis ser. israelensis. Using an immunoassay, the author’s labeled antibodies bound to cadherins and placed a number of these receptors on the membrane of apical and distal caecum larvae, precisely where the toxin is known to act. Currently, it is believed that cadherin is the major cytotoxicity mediator of Bt in Lepidoptera. Synthesis of membrane transporters Another mechanism of resistance to Bt toxins results from genes that synthesize membrane elements that require the ABCC2 transporter. These conveyors are normally associated with drug resistance in eukaryotic cells and are known to carry the chemicals out the cytosol. Gahan et al. (2010) investigated the resistance of Heliothis virescens (which affects cotton crops) to Cry1Ac. In vitro tests indicated the presence of a receptor that binds cadherins, but field tests indicated that there was resistance. Finally, genetic analysis showed the existence of the ABCC2 transporter membrane, which minimizes the toxic action of Bt. Baxter et al. (2011) studied the resistance of Plutella xylostella (Lepidoptera: Plutellidae) and Trichoplusia ni (Lepidoptera:Noctuidae) to the Cry1Ac gene, and after extensive genetic mapping, they verified the presence of carriers of ABCC2. The authors detected the presence of five resistance genes at the same locus on chromosome 15. These genes play a crucial role in the resistance of cells to toxins by removing the active form of the toxin from the cytosol and inhibiting the cytotoxic action of Cry1Ac. Similar results were observed by Hernańdez-Rodriguez et al. (2012), who also found that resistance genes harbored the ABCC2 transporter, which aids in the resistance of P. xylostella. Transregulatory mechanism Unlike the other research, the study by Tiewsiri & Wang (2011) found no change in the receptor function or the

Genes for resistance to Cry1Ac carriers include ABCC2 transporters While APN1 undergoes downregulation, APN-6 is upregulated

Tiewsiri & Wang (2011)

membrane transporters that expel the toxin from the cytosol. When evaluating resistant populations of Trichoplusia ni (Lepidoptera: Noctuidae), the authors found the so-called transregulatory mechanism. Two N-aminopeptidase receptors, APN1 and APN6, are produced in the same proportion in a susceptible cell, and this regulates its balance. When the production of APN1 was reduced significantly, the production of APN6 increased. This prevented the activity of the toxin, which then resulted in cytotoxic inactivity and resistance. The reasons for the imbalance are not clear, but it is believed that when the synthesis of APN1 decreased, APN6 increased as a compensatory mechanism. Regardless of the reasons, changes in the transregulatory mechanism may inhibit the activity of Cry1Ac without requiring changes in the structure of the receptors. Resistance management Over the years, there have been an increasing number of reports of insect resistance to both Bt insecticides and to transgenic plants that produce the Cry toxin. A pyramid strategy has been attempted where the cultivates receive genetic material from two different toxins in the hope that this will reduce the survival of resistant organisms. This strategy was adopted for cotton plants affected by a species of Lepidoptera (Tabashnik et al., 2009). Initially, genes were inserted so that the plant produced Cry1Ac, and then genes were inserted so that Cry2Ab was also produced. This was an efficient strategy for preventing the development of resistance because it involves toxins that have different binding sites; this prevents cross-resistance. However, in practice, what was observed did not exactly match the theoretical prediction, and this demonstrated that resistance may follow different patterns. In a study of cotton cultivars affected by Pectinophora gossypiella (Lepidoptera: Gelechiidae), Tabashnik et al. (2009) observed asymmetrical cross-resistance. The authors found that individuals with resistance to Cry1Ac also had a high rate of resistance to Cry2Ab. On the other hand, the reverse did not occur: resistance to Cry2Ab did not predict resistance to Cry1Ac. The authors suggest that resistance to Cry1Ac probably involves two or more gene loci, while resistance to Cry2Ab requires only one locus, which is shared by resistance to Cry1Ac. The emergence of insect resistance to Bt has become especially relevant for transgenic cultivars, and thus management strategies have been developed to eliminate or at least to reduce its incidence. For example, the use of refuge areas has


DOI: 10.3109/07388551.2014.960793

been studied as a way to combat resistance (Carrie`re et al,. 2004; Lenormand & Raymond, 1998). To follow this strategy, producers allocate an area that will be free of Bt insecticides and close to 20% non-transgenic cultivars. In this area, a diversity of insects sensitive to Cry is maintained; in this way, excessive proliferation of toxin-resistant individuals can be avoided. The advocates of this theory believed that this would reduce the frequency of alleles for resistance and extend the duration over which the toxin was effective (Lenormand & Raymond, 1998; Tabashnik et al., 2009). Although this method has been applied, the results are not conclusive. Apart from implementation difficulties, evaluation of the results has raised doubts about its efficiency. Carrie`re et al. (2012) found divergent results; i.e. certain cultivars produced a reduction in the frequency of resistant individuals, but in others, no difference was observed. After evaluating a large number of locations and cultivars, the authors concluded that an analysis of the spatial conditions of each environment was important in order to develop and implement such refuges; i.e. large and careful studies are needed to properly plan these refuges. Since the Bt mechanisms of action can vary and produce diverse cellular responses that culminate in cell death, invertebrates use multiple strategies to escape the action of the toxins. Although this field of research is new, different metabolic pathways that result in resistance to Bt have been detected, and probably many more are yet to be described. Studies leading to the understanding of these mechanisms are indispensable for the development of resistance management strategies and to ensure the effectiveness of biological insecticides and genetically modified crops. Resistance in Culicidae For decades, studies related to development of resistance B. thuringiensis involved only insects of the order Lepidoptera, whose species has been exposed to Bt products since the 1960s (Milner, 1994). After the sub-species israelensis isolation and its biological insecticides production, the use of the microorganism to control mosquitoes began over 20 years ago (Margalit & Dean, 1985). Thus, reports of resistance are much more recent in relation to the phenomenon observed in caterpillars. The resistance to Bt insecticide was reported in laboratory experiments with Culicidae species, with confirmation of efficiency reduction to the bacteria products. Goldman et al. (1986) found resistance in Aedes aegypti after the 14th generation through artificial selection of individuals. Georghiou & Wirth (1997) observed the same phenomenon in populations of Culex quinquefasciatus, decreasing the efficiency and increasing resistance, especially with the biological insecticide with Cry4D. Despite the strong evidence in laboratory experiments, fieldwork involving populations of mosquitoes that developed resistance naturally had not yet been confirmed (Bravo et al., 2007; Tabashnik, 1994). The development of resistance in the front of culicids of Cry toxin has been proven in the field only recently with some observed patterns. Wirth et al. (2012) discussed the evolution of the cross-resistance to Cry4Aa, Cry4Ba Cry11Aa toxins in


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Culex quinquefasciatus, featuring autosomal inheritance. Tetreau et al. (2013) monitored the emergence of Aedes sticticus tolerance (Meigen) to Cry4a and Cry11Aa after decades of biopesticide application. A similar result was verified by Stalinski et al. (2014), who studied the development of resistance in Aedes aegypti to the Bti, verifying that cross-resistance to the same toxins studied in Wirth et al. (2012), which indicates the existence of a resistance in the same locus activation site. The genetic mechanisms related to resistance were approached by Bonin et al. (2009), who investigated candidate genes to Bti resistance in Aedes aegypti. Comparing the genomes of susceptible and resistant strains, the authors identified genes that encode cadherin and peptidases enzymes, indicating that these peptides were related to resistance. Paris et al. (2012) did not study changes in genes but examined the degree of variation during transcription. The authors noted that resistant strains exhibit significantly more varied genes during the transcripts than the susceptible strains, indicating a strategy of resistance without the changing of genotype. The participation of cadherin is well known and has been studied in caterpillar resistance to Bt toxins. Another mechanism of resistance was analyzed by Tetreau et al. (2012), who found a lower expression of membrane Cry receptors in resistant strains of Aedes aegypti, also similar to Lepidoptera. Proteomic and transcriptomic analysis showed a reduction of receptors for toxins, preventing the events that culminate in the death of the larva. Boyer et al. (2012) observed the presence of detoxifying enzymes such as those responsible for resistance. Assessing the mortality of larvae and the enzymatic activity, the authors found a positive correlation between glutathione S-transferase and a lower mortality rate of larvae. Another approach to the mechanism of resistance in Culicidae was proposed by Patil et al. (2013a,b). The authors studied the relationship between larval gut microflora and Cry toxins, resulting in the development of resistance. According to this view, the presence of certain microorganisms in the larva midgut could result in a protective action because it could degrade the bacterial toxin before its activation. Thus, the presence of these microorganisms inhibits pathogenic action and prevents the events that culminate in cell death. This line of study ignores the role of changes of Cry receptors in resistance, enzyme mechanisms related to the tolerance of the larvae, and the genetic origin of resistance, but it demonstrates the complexity of the understanding of insect resistance to the biological insecticide.

Other applications of B. thuringiensis Nearly a century after the detailed description of Berliner, Bt has become an important tool in the control of pests that affect humans, whether in agriculture or in combating vectors of disease. Since the first commercial use of Bt formulations for the control of caterpillars, the spectrum of applications has increased and is no longer restricted to the initial function. It has become apparent that the potential of Bt transcends just biological control, and it can be used in other branches of science to assist humans.

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Chitinase production Chitinases are enzymes that degrade chitin, a polysaccharide composed of polymers that is widely distributed in nature. This enzyme can be found, for example, in the cell walls of fungi, the exoskeletons of arthropods, and the shells of crustaceans and nematodes (Dahiya et al., 2006). Chemically, chitinases act by hydrolyzing N-acetyl D-glucosamine, and this capability has great industrial potential. By having the affinity to the polymer and the ability to degrade it, chitinases have many applications in the field of health care for their antibacterial and immunomodulating effects and in agriculture for the production of antifungal substances and the control of plant pathogens (Bhattacharya et al., 2007; Reyes-Ramı´rez et al., 2004). The presence of chitinase enzymes in Bt was found only recently, after many decades of field use. It had usually been assigned a secondary role in the cytotoxicity of organic insecticides, since it was believed that the toxicity was due almost exclusively to the Cry toxins. However, recent studies have changed this view, giving due importance to the biological activity of bacterial chitinase (Sampson & Gooday, 1998; Wiwat et al., 2000). It is known that chitinases act synergistically with Cry, providing increased rates of mortality (Liu et al., 2002). These enzymes act by breaking the chitinous exoskeleton of an invertebrate, and thus aiding in the bacterial invasion of tissues and the establishment of septicemia before leading to death. Vu et al. (2009) discovered that when they used a media that cultured a high production of chitinases, even with fewer bacterial cells and a lower concentration of spores, they were able to obtain significantly higher toxicity than that achieved by cultivation in a media that produced a lower concentration of chitinase. Ramirez-Suero et al. (2011) estimated the synergistic effects of chitinase on the toxicity of various Bt isolates in mortality tests with A. aegypti. They found that the synergism had a factor of between 1.4 and 2, indicating the importance of these enzymes in the biological activity of the bacteria. Ever since it was shown that the production of chitinases increases the Bt biopesticide toxicity in biological assays, researchers have attempted to stimulate the production of this enzyme when producing biopesticides (Brar et al., 2008; Dahiya et al., 2006;). To accomplish this, it is necessary to add chitin to the medium, since this stimulates the bacteria to secrete chitinase to degrade the substrate; the chitinase then accumulates in the supernatant during fermentation. In some cases, when the main goal is the production of this enzyme, one can use chitin as the sole source of available carbon in the medium. In other studies, when toxicity is the goal, the polymer is only added to supplement the medium (Vu et al., 2009; Wang et al., 2006). Even when the production of chitinase is not a central concern in the formulation of a medium for growing Bt, enzyme production is often stimulated indirectly, primarily by the use of complex substrates. Chaiharn et al. (2013) showed satisfactory toxicity results using shrimp waste, including the chitinous exoskeleton, in the formulation of the medium for Bt. Likewise, Patil et al. (2013a,b) fermented bacteria and obtained high mortality outcomes using a medium formed


from the exoskeleton of the silkworm, for which close to 75% of the dry mass is chitin. As described by Melo et al. (2012), when growing Bt in poultry litter, the addition of chitin occurred indirectly. This occurred when the waste excreted by birds or by beetles in the larval or adult stage were added to the sawdust and wood shavings in the culture medium. In all three cases, the presence of chitin apparently stimulated the production of chitinase, which then increased the action of the Cry toxins produced by the Bt. Although chitinases stimulates increased toxicity of Bt products, its use in biopesticides is just one of the potential uses of these enzymes. In a study of soybeans, Reyes-Ramirez et al. (2004) investigated the protective, antifungal activity of chitinase from Bt. Fungi are a serious problem in agriculture, and they reduce the seed germination rate and inhibit the growth of plants. It is a challenge to find substances that protect the seeds. In that study, the authors treated the seeds with Bt chitinases and then inoculated them with spores of fungi. They noted germination rates of up to 93% for the soybeans that had been previously contaminated with the fungus Sclerotium rolfsii Sacc, attesting to the degrading effect of chitinase on the walls of the fungus. When evaluating the chitinase produced by B. thuringiensis ser. kurstaki on effluents with sewage sludge, Brar et al. (2008) observed the presence of chitinases with molecular weights ranging from 36 to 45 kDa; these were then analyzed by SDS-PAGE. The authors observed that the peak production of enzymes occurred between 15–30 h of fermentation, maintaining an activity rate of 96–99% after two weeks of incubation, with the temperature and pH controlled (4.0). These studies are essential for characterizing the use of these enzymes, and they demonstrate the potential for the production of chitinase by alternative means of fermentation with Bt. These studies indicate that the production of chitinases is an important variable in the toxicity of Bt products. When the exclusive goal of fermentation is the production of Bt spores and the Cry toxins, it is important to consider efficiency. We note that the production of chitinases can lead to another application of Bt: control of fungi that are not susceptible to the Cry toxin. BT activity against cancer cells The entomopathogenic properties of Bt are known and widely applied; however, it is important to remember that most strains of bacteria do not have this property and are innocuous to invertebrates (Ohba et al., 1988). Despite producing toxins and eliminating them during sporulation, biological activity and cytopathic damage are not observed in some organisms that have been tested. For a long time, this fact has intrigued scientists, who began testing possible applications of the protein content of these non-toxic strains. A surprising observation was made by Mizuki et al. (1999), who reported a new category of Bt proteins with molecular weights that were less than those of the Cry toxins and that did not cause cytopathic damage to the intestinal epithelium of invertebrates but did show toxic effects on the altered cells of some types of cancer. These toxins with molecular weights below 90 kDa were named parasporins. These peptides showed toxic effects on


DOI: 10.3109/07388551.2014.960793

7

Table 2. Known types of parasporins, their biological activities, and published studies.


Parasporin

Mode of cytotoxic action

Susceptible cell line 2+

PS-1

Induction of apoptosis and "[Ca ]

PS-2

Pore formation and osmotic imbalance

PS-3

Not investigated

PS-4

Pore formation and osmotic imbalance

HeLa HCT-250 HepG2 HeLa HL60 HepG2 Molt-4 CACO-2 Sawano

cancer cells in a discriminatory manner; i.e. affecting only the changed cells and causing no damage to normal cells. So far, four types of parasporins have been described: PS-1, PS-2, PS-3 and PS-4 (Table 2). Their biological activities have been studied but have yet to be fully elucidated (Mizuki et al., 1999). A characteristic common to all parasporins is the need for activation in order to become biologically active. This process can be done enzymatically by treatment with proteinase K and trypsin in an alkaline environment or by another method. This can be compared to the activation of entomopathogenic toxins, which require the action of digestive proteases in the alkaline environment of the digestive tract of invertebrates before they can trigger cytopathic damage. Among the parasporins, the two that have been the most studied are PS-1 and PS-2. The activity of PS-1 was documented by Katayama et al. (2005), who observed different degrees of cytotoxicity in four strains of the cells derived from cancerous tissues; in particular, HeLa cells from human cervical carcinoma showed an increased susceptibility to the activated toxin. Poornima et al. (2010) investigated a total of 82 native isolates of strains of Bt that produce parasporin-1, which is toxic to colon cancer cells (HCT-250) and lymphoma (U-937). One strain of Bt had high cytotoxicity for the HCT-250 cells type but only medium cytotoxicity for the U-937 cells. Nagamatsu et al. (2010) analyzed the cytopathic damage in cells of liver cancer (HepG2) and cervical cancer (HeLa) caused by PS-1 and observed morphological changes such as cell swelling and vacuoles in the cytoplasm, similar to those observed during apoptosis. Cytopathic damage was also studied by Kitada et al. (2006) but with PS-2 in liver cancer cells (HepG2). These authors found alterations in the cytoskeleton, fragmentation of the mitochondrial and endoplasmic reticulum, and an abnormal increase in cell permeability. Abe et al. (2008) studied the PS-2 in cells of liver cancer and verified the oligomerization of the pore-forming toxin that bound to lipids in the plasma membrane. Akiba et al. (2009) analyzed the structure of the formation of pores in the plasma membrane while researching HepG2 with a PS-2 extract. The authors found the presence of three domains, the first responsible for binding and the last two responsible for pore formation. Yamashita et al. (2005) investigated the biological activity of PS-3 applied to 16 cell lines, including nine cancerous tissues, four intact human tissues, and three normal mammalian cell lines. The authors showed the toxic effects of PS-3 on HeLa and HL60 cell lines of myeloid leukemia and cervical cancer, respectively. However, the mechanisms of cytotoxicity

Type of cancer

References

Cervical cancer Human Colon cancer Human Liver cancer Cervical cancer Myeloid leukemia Human Liver cancer Lymphocytic leukemia Adherent colon cancer Endometrial cancer

Katayama et al. (2005) Poormina et al. (2010) Kitada et al. (2006) Akiba et al. (2009) Yamashita et al. (2005) Okumura et al. (2011)

were not investigated in this study. Okumura et al. (2011) studied the cytotoxicity of PS-4 and reported on their cytotoxicity on CACO-2, Sawano and Molt-4 cell lines, corresponding to cells of colon cancer, endometrial carcinoma and lymphoblastic leukemia, respectively. They observed typical morphological changes of osmotic stress, such as swollen cells and shrinking cores, and noted a crash after 24 h. The authors examined the binding of pores formed in the plasma membrane through activation of the cholesterol molecules. Parasporins 1 and 2 were the focus of a study by Ohba et al. (2009), who found different models that result in the death of abnormal cells. In the first, PS1a1, there was a rapid increase in the intracellular concentration of Ca2+ but no increase in the permeability of the membrane. The authors concluded that the parasporin activated the mechanism of apoptosis in susceptible cells. Moreover, PS2a1 does not act in the cytoplasm but increases the permeability of the membrane of cancer cells. This leads to an osmotic imbalance and is the result of binding with the receptor site and oligomerization of the toxin to form the pre-pore. Once coupled to the lipid bilayer, these structures result in the loss of membrane function and death of the cell. These results indicate that the mode of action of PS-1 and PS-2 are very similar to the pre-existing toxicity models for cells in the digestive tract of invertebrates. As with the model, cell damage is caused by the formation of pores and the loss of membrane integrity, and PS-2 also forms oligomers that bind to the lipid plasma membrane, resulting in pores that result in death. Exactly as observed in the signaling pathway, where toxins are recognized by cadherin receptors and activate cell death mechanisms involving channels of Mg+2, PS-1 appears to activate apoptosis by increasing concentrations of intracellular Ca+2. In other words, despite the differences in the various cells studied, very similar processes are capable of causing the death of digestive tract cancer cells of both invertebrates and vertebrates. After discovering the distinct mechanisms by which parasporins can cause pore formation or induce apoptosis, an interest then began to develop a microorganism that could simultaneously produce two or more types of parasporins. This was achieved by Okumura et al. (2013), who cloned the gene of PS-2, inserted it into a plasmid, and added it into a bacteria that then naturally produced PS-4. Tests with Escherichia coli confirmed the presence of the genes on the plasmid and that the parasporins were expressed simultaneously.


8


Although the mechanism of the cytotoxic action of parasporins is not yet completely understood, there is growing interest in the application of these toxins as a possible treatment for cancer. Wong (2009) evaluated the occurrence of competition between active sites between parasporins and commercial anti-cancer drugs. Using confocal microscopy to analyze the binding sites, which were labeled by biotin, the authors showed that there were few sites in common, attesting to a low competition (530%), but no significant effects. This demonstrated that Bt parasporins have the potential to be a new tool in the treatment of cancer cells, and that they can be used without interfering with the current therapeutic treatments. It is undeniable that parasporins have the potential to develop new anti-carcinogenic substances. However, it is important to note that the majority of the results were performed with in vitro tests, which only simulate biological systems. In one of the few in vivo studies available, El-Hag & Safti (2011) tested the strain B. thuringiensis ser. dakota for its ability to fight cancer cells in mice. The authors observed a significant increase in the survival of treated rodents compared to the control group. Likewise, the number of metastases decreased and even disappeared in some cases. According the authors, this increased survival time could be attributed to greater resistance to tumors in the treated mice. These results point to the potential of B. thuringiensis parasporins as a source of possible new drugs for fighting cancer.

Summary

The cytotoxicity of Cry proteins in the digestive tract of invertebrates may involve the formation of pores, an osmotic imbalance and/or the stimulation of apoptosis. Resistance to Bt grows on Lepidoptera and Culicidae, caused by flaws in the receipt of toxins, caused by a change in the cadherin receptor, the formation of membrane transporters and/or altered regulation of the synthesis of receptors. The production of chitinase by Bt is an important variable in the efficiency of insecticides produced by bacteria; it acts synergistically with the Cry toxins and increases the efficiency of the formulations. Parasporins can produce a cytotoxic effect on certain kinds of cancer cells by producing pores or stimulating apoptosis, which is similar to what occurs in the susceptible cells of invertebrates.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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