Heliothis virescens and Manduca sextaLipid Rafts Are Involved in Cry1A Toxin Binding to the Midgut Epithelium and Subsequent Pore Formation

Zhuang, Meibao; Oltean, Daniela I.; Gómez, Isabel; Pullikuth, Ashok; Soberón, Mário; Bravo, Alejandra; Gill, Sarjeet S.

doi:10.1074/jbc.m110057200

Cited by 157 publications

(140 citation statements)

References 67 publications

(68 reference statements)

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“…The interaction of the monomeric Cry1A with the cadherin receptor induces toxin oligomerization [4,5]. The oligomerization of Cry1A toxin increases its affinity to the second receptor, aminopeptidase-N. Aminopeptidase-N localizes the toxin in membrane microdomains, where it is inserted and induces the formation of ionic pores [4,6]. These ionic pores shunt the potential difference gradient and disrupt the K + and H + gradients, affecting nutritional uptake with eventual lysis of the midgut cells [2].…”

Section: Introductionmentioning

confidence: 99%

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

et al. 2006

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Abstract. The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K + permeability. A fluorometric assay performed with 3,3¢-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K + -selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

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Section: Introductionmentioning

confidence: 99%

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

et al. 2006

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“…Also, they function as pathogen portals for different viruses, bacteria and toxins [7,38]. Recently, we demonstrated that the APNs in M. sexta and H. virescens, are located in lipid rafts, and that the integrity of these microdomains is essential for Cry1Ab pore activity [46]. These data could indicate that signal transdution induced by Cry toxin insertion into lipid rafts could be part of the mechanism of action of these toxins, but this remains to be demonstrated.…”

Section: Final Remarksmentioning

confidence: 97%

“…For many years the Cry toxins have been recognized as pore-forming toxins that induce cell death by forming ionic pores [10,35,39]. Cry toxins insert into lipid rafts membranes present in the apical microvilli membrane of midgut cells of susceptible larvae, causing osmotic lysis and subsequent death of the larvae [3,10,39,46]. A second model proposed that these toxins activate a signal pathway through receptor interaction, resulting in insect cell death without the participation of lytic-pores [45].…”

Section: Models Of the Mode Of Action Of Cry Toxins In Lepidopteran Imentioning

confidence: 99%

“…The oligomeric structure has been observed in several Cry toxins [1,13,18,26,30,33,36,37,40]. The pre-pore binds to a second receptor, the GPI-anchored aminopeptidase, due to its higher affinity to this receptor [3], facilitating the insertion of the oligomer into membrane lipid rafts and forming pores [3,46]. Importantly, another GPI-anchored protein, an alkaline phosphatase, has been recognized as a Cry1Ac receptor in Heliothis virescens and M. sexta [22,23].…”

Section: Models Of the Mode Of Action Of Cry Toxins In Lepidopteran Imentioning

confidence: 99%

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The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

et al. 2008

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The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion.For formation of these oligomers coiled-coil structures are important, and helix α-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix α-3 are affected in pore formation and toxicity. Mutants affected in toxicity bind Bt-R 1 receptor with a similar K D as the wild type toxin but do not form oligomers nor induce pore formation in planar lipid bilayers, indicating that the pre-pore oligomer is an obligate intermediate in the intoxication of Cry1Ab toxin and that interaction of monomeric Cry1Ab with Bt-R 1 is not enough to kill susceptible larvae.

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“…The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3,4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1,5). Although this mechanism of action is generally accepted, it may involve additional binding molecules, such as glycolipids, or more than one glycosylphosphatidylinositol-anchored receptor (5,6).…”

mentioning

confidence: 99%

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

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Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

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Heliothis virescens and Manduca sextaLipid Rafts Are Involved in Cry1A Toxin Binding to the Midgut Epithelium and Subsequent Pore Formation

Cited by 157 publications

References 67 publications

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

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