Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins

Abstract

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound ∼60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of ¹²⁵I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K_d∼0.1 μM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.

Introduction

Innate cellular defense involves nonspecific responses found in organisms ranging from plants to humans. Invertebrates, including insects, rely primarily on innate cellular responses to defend themselves against microorganisms and associated toxins (Hoffmann et al., 1996, Hoffmann et al., 1999). Information relay between innate defenses and adaptive protective immunity are of considerable scientific interest and insects serve as useful model systems to investigate the molecular and biochemical pathways associated with these conserved responses. For example, since the discovery of antimicrobial activities in the cecropia moth, Hyalophora cecropia (Steiner et al., 1981), studies of Drosophila and mosquitoes have revealed notable conservation of innate defense mechanisms in both insects and mammals (Hoffmann et al., 1999, Dimopoulos et al., 2001). These immune responses are triggered on the surface of epithelial cells and communication through signaling pathways promotes special physiological adaptations that somehow protect the cell against foreign invaders. Most often, these reactions sustain cellular adaptation upon prolonged exposure to microbial activity or other stress conditions and eventually lead to resistance and, in some instances, apoptosis (Hecht, 1999, Loeb et al., 2000).

Commercial formulations of Bacillus thuringiensis, which produces insecticidal crystalline proteins known as Cry toxins are utilized to control many agriculturally and biomedically important insects (Schnepf et al., 1998). The toxicity as well as specificity of B. thuringiensis Cry toxins correlates directly with binding of toxins to high-affinity receptors on the epithelial cells that line the midgut of susceptible insects. Cry toxin receptors in the midgut epithelium of certain insects, including Manduca sexta and Bombyx mori (Vadlamudi et al., 1993, Vadlamudi et al., 1995, Nagamatsu et al., 1998, Nagamatsu et al., 1999) were identified as specific cell adhesion molecules, cadherins, which represent a large family of calcium-dependent, transmembrane glycoproteins and are responsible for maintaining the integrity of cell–cell contacts in multicellular organisms (Nollet et al., 2000, Angst et al., 2001). In addition to cadherin receptors for Cry toxins, several 120-170kDa Cry toxin-binding proteins also have been identified in some lepidopteran insects (Knight et al., 1994, Knight et al., 1995, Sangadala et al., 1994, Valaitis et al., 1995, Gill et al., 1995, Oltean et al., 1999). These proteins, in fact, exist as multiple forms of a ubiquitous midgut protease, aminopeptidase N (APN). Although APN interacts with Cry toxins and is an important component of the insect midgut surface, APNs do not serve as functional receptors to mediate toxicity of Cry proteins. In contrast, binding of Cry toxins to the cadherin receptors in the insect midgut causes stress and, consequently, disrupts the epithelium and destroys the entire midgut tissue. Insect larvae that have ingested lethal amounts of the Cry toxin stop feeding and die. In M. sexta, the cadherin receptor for Cry1A toxins, BT-R₁, is specifically expressed in the midgut of the larval stage of the insect (Midboe et al., 2002). That the molecule is not present in any other stage of the insect's life cycle indicates that Cry toxin binding to developmentally important cadherins might be of evolutionary significance to the entomopathogenicity of B. thuringiensis. Furthermore, cadherins are implicated in resistance of insects to the insecticidal activity of Cry toxins. Resistance to the Cry1Ac toxin by Heliothis virescens is linked to retrotransposon-mediated disruption of a specific cadherin gene (Gahan et al., 2001), indicating that midgut epithelial cadherins are involved directly in the entomopathogenicity of B. thuringiensis. Of particular concern is the likelihood that insect resistance to certain Cry toxins will become prevalent (Ferre et al., 1995, Schnepf et al., 1998).

Little is known about the mechanism(s) of resistance to Cry toxins. One possible mechanism includes decrease in the toxin binding to insect midgut (Van Rie et al., 1990, Ferre et al., 1991, Ferre et al., 1995, Sayyed et al., 2000). Others implicate proteases that interact with toxin in the insect gut. For example, proteases from a strain of tobacco budworm H. virescens resistant to B. thuringiensis subsp. kurstaki HD-73 were reported to degrade toxin faster than proteases from a susceptible strain (Forcada et al., 1996). Keller et al. (1996) showed that the specific activity of gut proteases increases throughout larval development. The increased activity was associated with a loss of sensitivity by late developmental stages of the larvae to Cry1C toxin, possibly, due to toxin degradation. Oppert et al. (1997) reported two resistant strains of the Indianmeal moth Plodia interpunctella that lacked a major gut protease involved in toxin activation. These investigations suggest that changes in the activity or composition of gut proteases are involved in altering the susceptibility by insects to Cry toxins.

In the present study, we assessed several factors that are implicated in resistance by the CPB to the Cry3Aa toxin produced by B. thuringiensis subsp. tenebrionis. We compared proteolytic activity profiles in BBMV and gut juice, aminopeptidase activity, stability of Cry3Aa toxin to BBMV and gut juice proteases and toxin binding to BBMV as well as toxin action on midgut epithelial cells in vivo. We found that resistance to Cry3Aa toxin entails decreased toxin binding and changes in the composition and activity of midgut proteolytic enzymes, especially elevated aminopeptidase activity. These factors appear to be involved in maintaining a protective state in midgut epithelial tissue, and, together, they constitute an adaptive response of the CPB to the Cry3Aa toxin.