Dominant Negative Mutants of Bacillus thuringiensis Cry1Ab Toxin Function as Anti-Toxins: Demonstration of the Role of Oligomerization in Toxicity

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155

157

Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K d ‫؍‬ 0.6 nM), whereas the monomer showed weaker binding (apparent K d ‫؍‬ 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in ␤-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.The Cry toxins produced by Bacillus thuringiensis are used worldwide as effective biological control agents for many species of insects including agricultural and forest pests and several vectors of human and animal diseases. Its insecticidal property results from crystalline inclusions produced during sporulation that are formed by ␦-endotoxins known as Cry toxins (1).The Cry protein family is composed of more than 54 groups, among which the three-domain Cry family forms the major group, having members that show toxicity to different insect orders and to nematodes. The crystal structure has been solved for six three-domain Cry toxins and one protoxin that display different insect specificities (2). The activated Cry toxins are globular molecules consisting of a bundle of seven ␣-helices (domain I), a three-␤-sheet prism (domain II), and a ␤-sandwich (domain III) (3, 4). The N-terminal domain I is involved in oligomer formation, membrane insertion, and pore formation (5-8). Domain II and III are involved in the recogn...

Section: Discussionmentioning

confidence: 99%

Role of Alkaline Phosphatase from Manduca sexta in the Mechanism of Action of Bacillus thuringiensis Cry1Ab Toxin

Arenas

Bravo

Soberón

et al. 2010

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157

“…Dominant-negative inhibitors of VacA (15)(16)(17)(18)(19), protective antigen (20 -25), Cry1Ab (26), and ClyA (27) are believed to act through the formation of mixed oligomers containing both wild-type and mutant proteins. Direct evidence of mixed oligomers, based on Förster resonance energy transfer or co-immunoprecipitation, has been reported in a few studies (16,18,19,63).…”

Section: Discussionmentioning

confidence: 99%

“…Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins are mixed together. Such dominant-negative inhibitors have been described for a diverse set of toxins, including Helicobacter pylori VacA (15)(16)(17)(18)(19), Bacillus anthracis anthrax toxin protective antigen (20 -25), Bacillus thuringiensis Cry1Ab (26), and Escherichia coli ClyA cytotoxin (27). Like VacA, protective antigen, Cry1Ab, and ClyA, the ⑀-toxin assembles into oligomeric complexes containing multiple toxin monomers (28 -30).…”

mentioning

confidence: 99%

Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

Pelish

McClain

2009

The Clostridium perfringens ⑀-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ⑀-toxin. Site-specific mutations were introduced into the gene encoding ⑀-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wildtype ⑀-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ⑀-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric poreforming toxins and provide the basis for developing new therapeutics for treating intoxication by ⑀-toxin.The Clostridium perfringens ⑀-toxin is one of the most potent bacterial toxins (1, 2). The ⑀-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3-6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7-9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ⑀-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10 -13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ⑀-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins a...

“…of Cry1A toxins, supported that helices ␣-3, ␣-4, and ␣-5 are important for toxicity and pore formation, without affecting their binding interaction with toxin receptors (11)(12)(13)(14)(15). The helix ␣-3 participates in the oligomerization process (13), and it is proposed that the hydrophobic hairpin formed by helices ␣-4 and ␣-5 inserts into the phospholipid bilayer whereas the rest of amphipathic helices of domain I, excluding helix ␣-1, are spread on the membrane surface in an umbrella-like conformation (10, 16 -19).…”

mentioning

confidence: 95%

Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Remain Exposed to the Solvent after Insertion of Part of Domain I into the Membrane

Zavala¹,

Pardo-López²,

Cantón

et al. 2011

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Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of ␣-helices 4 -5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membranebound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.