Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

Abstract

<jats:p>Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7–11 (cadherin repeats 7–11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7–11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7–11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7–11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.</jats:p>

Aedes cadherin mediates the in vivo toxicity of the Cry11Aa toxin to Aedes aegypti

“…This conformational change is thought to cause the formation of oligomers that bind to a secondary receptor, which can be APN or ALP anchored to the membrane by a GPI anchor [6, 17]. Similarly, in Aedes mosquitoes, either Cry11Aa protoxin in the presence of BBMV or proteolytically activated Cry11Aa toxin incubated with AaeCad receptor fragment (CR7-11) alone can trigger toxin oligomerization [36, 38]. Here we also showed that C6/36 cells expressing full-length AaeCad can cause Cry11Aa oligomerization by the use of trypsin-activated Cry11Aa toxin directly or Cry11Aa protoxin in the presence of midgut juice (Fig.…”

“…Although AaeCad RNAi-mediated silencing had been done in Aedes mosquitoes by dsRNA feeding [38], a transgenic Aedes mosquito line with silenced AaeCad was established in order to obtain more homogeneous animals that can be used to screen a number of other toxins. Importantly, these transgenic mosquitoes revealed a higher tolerance to Cry11Aa (about 13 fold) at LC 50 value than Aedes larvae fed AaeCad dsRNA, where only a 50% reduction in Cry11Aa toxicity was obtained compared to wild-type mosquitoes [38].…”

“…Importantly, these transgenic mosquitoes revealed a higher tolerance to Cry11Aa (about 13 fold) at LC 50 value than Aedes larvae fed AaeCad dsRNA, where only a 50% reduction in Cry11Aa toxicity was obtained compared to wild-type mosquitoes [38]. …”

Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti

“…aegypti (Nene et al, 2007). Previous studies investigated three ALPs as a receptor of Bti Cry toxins and found that ALP1 (AAEL009077) is a functional receptor of Cry11Aa and Cry4Ba (Fernandez et al, 2009; Rodríguez-Almazán et al, 2012). In transcriptome analysis, AAEL013330 and AAEL015070 are likely putative Cry11Aa receptors, since both of these ALPs were significantly reduced in the resistant larvae midgut.…”

“…These proteins are also present in the posterior midgut epithelial cells and/or the gastric caeca (Chen et al, 2009b; Chen et al, 2013). RNAi-mediated silencing of cadherin and of APN (AAEL012783) increased tolerance for Cry11Aa (Lee et al, 2014; Rodríguez-Almazán et al, 2012) and Cry4Ba toxicity (Saengwiman et al, 2011), respectively. These data collectively suggest that cadherins and APNs may be functionally important for Cry toxicity in mosquitoes.…”