Mode of Action of Cry Proteins

Owing to their widespread occurrence and importance to the efficacy of Bt insecticides used in agriculture, forestry, and vector control, Cry proteins have been the subject of numerous mode of action studies over the last two decades. Prior to this, it was known that Cry proteins are not contact poisons (as are most synthetic chemical insecticides) but, rather, are insecticidal proteins that act on the midgut and, being proteins, must be ingested to be effective. It was also known that these proteins had to be cleaved by midgut proteases to be active — cleavage releases the active toxin, which then binds to specific receptors on the microvilli of the target insect's midgut epithelium (stomach). If the appropriate receptors are not present, there is little if any binding and thus toxicity.2 These studies, in combination with resolution of the three-dimensional structure of several Cry proteins,20,21 have provided the following basic understanding of the mode of action Cry proteins produced by Bt and have informed the construction insect-resistant crops.

Analysis of cry gene sequences combined with the three-dimensional structures of Cry3A, Cry1Aa, and Cry2A showed that the active portion of Cry toxins is a wedge-shaped molecule of three domains (Figure 3.3), and typically consists of approximately 600 amino acids (residues 30-630).20,21 The active toxin contains five blocks of conserved amino acids distributed along the molecule, and a highly variable region within Domain II. This is the primary region responsible for the insect spectrum of activity, as demonstrated through domain-swapping studies.22 The sensitivity of a specific insect species to a particular Cry toxin is directly correlated with the number and affinity of binding sites on the midgut microvillar membrane.23,24

Resolution of Cry3A crystal structure20 showed that Domain I of this protein is composed of amino acids 1-290 and contains a hydrophobic, seven-helix amphipa-thic bundle, with six helices surrounding a central helix. This domain contains the first conserved amino acid block and a major portion of the second conserved block. Theoretical computer models of the helix bundle show that after insertion

FIGURE 3.3 Illustration of the three-dimensional structure of Cry3A, the first Cry protein for which the structure was solved. The molecule consists of three major domains. Domain I is the pore-forming domain that results in destruction of midgut epithelial cells after insertion into midgut cell microvilli. Domain II functions as a binding domain, allowing the activated protein to bind to midgut microvilli when appropriate receptors are present on microvilli. Domain III also has binding subdomains, and adds structural stability to the molecule.

S-Endotoxin from B. thuringiensis

Domain I

S-Endotoxin from B. thuringiensis

Domain I

FIGURE 3.3 Illustration of the three-dimensional structure of Cry3A, the first Cry protein for which the structure was solved. The molecule consists of three major domains. Domain I is the pore-forming domain that results in destruction of midgut epithelial cells after insertion into midgut cell microvilli. Domain II functions as a binding domain, allowing the activated protein to bind to midgut microvilli when appropriate receptors are present on microvilli. Domain III also has binding subdomains, and adds structural stability to the molecule.

Domain III

and rearrangement, aggregations of six of these domains likely form a pore through the microvillar membrane.20,2' Domain II extends from amino acids 29l-500 and contains three antiparallel P-sheets around a hydrophobic core. This domain contains most of the hypervariable region and most of conserved blocks 3 and 4. The crystal structure of the molecule, together with recombinant DNA experiments and binding studies, indicate that the three extended loop structures in the P-sheets are responsible for initial recognition and binding of the toxin to binding sites on the microvillar membrane.25

Domain III is composed of amino acids 50l-644 and consists of two antiparallel P-sheets, within which are found the remainder of conserved block 3 along with blocks 4 and 5. The Cry3A structure indicated that this domain provides structural integrity to the molecule.20 More recent site-directed mutagenesis studies of conserved amino acid block 5 in the Cryl molecules show that this domain also plays a role in receptor binding and pore formation.^

To cause toxicity after activation, Cry proteins must cross the peritrophic membrane and bind to proteins on the surface of midgut microvilli before they can insert to form a pore. The first proteins identified as receptors in the mid-l990s were aminopeptidases.26 These extended into the midgut lumen but were tethered to the microvillar membrane. Subsequently, other molecules (including cadherins and glycolipids) were also shown to be midgut receptors for Cry proteins.2l Studies of these receptors showed that even more important than the type of protein or lipid receptor was the surface glycosylation on these, which provides the specific surface sugars that the Cry molecule recognizes and binds to. Importantly, recent studies have shown that invertebrates, but not vertebrates, have a glycosylating enzyme, BL2, which creates the specific sugar residues on the glycolipid microvillar receptor recognized by Cry proteins.27 The lack of this enzyme in vertebrates provides a possible explanation for why activated Cry proteins do not appear to bind to cells lining the stomach and intestines of vertebrates.28

Just prior to entry or immediately after, individual Cry molecules oligomerize, forming a complex of from four to six molecules that form the actual pore.29,30 Based on a variety of evidence, this pore is thought to be a cation-specific channel.30 Once a sufficient number of these channels have formed, a surplus of cations (e.g., K+) enter the cell. This causes an osmotic imbalance within the cell, and the cell compensates by taking in water. This process, referred to as colloid-osmotic-induced lysis, continues until the cell ruptures and exfoliates from the midgut microvillar membrane.30 When a sufficient number of cells have been destroyed, the midgut epithelium loses its integrity. This allows the alkaline gut juices and bacteria to cross the midgut basement membrane, resulting in death, the latter caused by B. thuringiensis bac-teremia and tissue colonization in lepidopteran species. In mosquito and black fly larvae, midgut bacteria do not cross the midgut epithelium until after death; thus, in these the cause of paralysis and death is apparently due only to the insecticidal Cry and Cyt proteins.

This overview of toxin structure, receptors, and binding requirements constitutes a series of steps that account for the specificity and safety of Bt insecticides and Bt crops, as summarized below.

1. Endotoxin crystals must be ingested to have an effect. This is one of the reasons why sucking insects and other invertebrates such as spiders are not sensitive to Cry proteins used in Bt insecticides or Bt crops.

2. After ingestion, Bt endotoxin crystals active against lepidopterous insects must be activated. Activation requires that crystals dissolve. This typically occurs in nature under alkaline conditions, generally in digestive juices in the midgut lumen, where the pH is 8 or higher. Most nontarget invertebrates have neutral or only slightly acidic or basic midguts. Under the highly acidic conditions in stomachs of many vertebrates, including humans, Cry and Cyt protein crystals may dissolve, but once in solution they are rapidly degraded to nontoxic peptides by gastric juices, typically in less than two minutes.

3. After dissolving into midgut juices, Cry proteins must be cleaved by mid-gut proteases at both the C-terminus and N-terminus to be active.31

4. Once activated, the toxin must bind to glycoprotein or glycolipid receptors on midgut microvillar membrane. Most chewing insects that ingest toxin crystals, even those with alkaline midguts (including many lepidopter-ans), do not have the appropriate receptors and thus they are not sensitive to activated Cry proteins. This is because the activated Cry molecule typically requires a specific arrangement of sugar residues on the receptor to bind effectively. As a result, even insects sensitive to one class of Bt proteins, such as larvae of lepidopteran species sensitive to Cry1 proteins, are not sensitive to Cry3 proteins active against coleopterans — they lack receptors for these. A high degree of specificity is even apparent within each order of sensitive insects. For example, larvae of Heliothis virescens are highly sensitive to Cry1Ac (hence its use in Bt cotton), but larvae of Spodoptera species, such as the beet armyworm (S. exigua) and fall armyworm (S. frugiperda) are typically insensitive to this protein at rates encountered in nature or when treated with Bt insecticides. Cry1Ac is activated in these insensitive species, but binding to receptors is inefficient. Of relevance to vertebrate safety, no significant binding of Cry proteins has been detected in mammalian stomach epithelial cells.28

5. After binding to a midgut receptor, the toxin must enter the cell membrane and form a cation-selective channel. This requires a change in the conformation of the active Cry molecule and oligomerization to form the channel.30

With respect to Level 5, at present the specific conformational changes and details of the oligomerization process that must take place to exert toxicity are not known. It is known, however, that high-affinity, irreversible binding can occur in some insects yet not lead to toxicity. This implies that a specific type of processing, i.e., another level of specificity, may be required for toxicity that occurs as or after the toxin inserts into the membrane.

In Bt crops, only a portion of the second level (i.e., Level 2) of the first five levels of specificity has been circumvented. When synthesized in plants, full-length and truncated Cry proteins do not form crystals, and even if quasicrystalline inclusions do form, most of the toxin synthesized remains in solution within the plant cells. Nevertheless, whether produced in plants as full-length or truncated protoxins, Cry proteins must still be properly activated after ingestion, that is, cleaved properly at the C- and N-termini. In some crops, plant proteases may activate the toxin. Nevertheless, even if activated, the toxin must meet the criteria for binding and membrane insertion defined above by Levels 4 and 5 to be toxic. Furthermore, with the one exception of Cry9C (which was engineered to resist rapid proteolytic cleavage), most Bt proteins produced in Bt crops are degraded rapidly under conditions that mimic the mammalian digestive system. Although it is still possible that a small amount of activated toxin may survive in the vertebrate stomach, there is no evidence that this would lead to toxic or allergic reactions. Thus, most of the inherent levels of specificity that account for the safety of Cry proteins used in commercial bacterial insecticides apply to these same proteins when used to make Bt crops resistant to insects.

Another important aspect of specificity and safety is the route by which an organism is likely to encounter a toxin. Even though pulmonary (inhalation) and intraperitoneal injection studies are done with microbial Bt insecticides and proteins, their normal route of entry by target and nontarget organisms is by ingestion. This is equally true for Cry proteins produced in Bt crops. Most nontarget insects are not feeding on the plant or plant exudates, and therefore they are not exposed to the Cry protoxins or the activated toxin. And even then, many insects that feed on Bt crops, such as aphids and white flies, are not exposed to any significant level of toxin, as these feed primarily through the vascular tissues, which contain little if any Cry protein toxin. In comparison to most synthetic chemical insecticides, which as contact poisons kill many nontarget organisms when used in any crop, forest, or aquatic ecosystem, Cry proteins used in Bt insecticides and Bt crops are inherently much safer due to their specificity and targeted dissemination in the environment.

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