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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2013)

Cadherins play pivotal roles in the toxicity of Bacillus thuringiensis Cry proteins. Here I tested and showed that an Aedes cadherin (AAEL007478) and an N-cadherin (AAEL000597) are involved in the in vivo toxicity of Cry11A toxin to Aedes aegypti. Aedes cadherin was stably expressed in a mosquito cell line and these cells showed increased sensitivity (37% death) to Cry11Aa toxin. These results show Aedes cadherin mediates Cry11Aa toxicity, but since high toxicity was not obtained, an additional secondary receptor may be needed for manifestation of full toxicity. Using a whole genome screen to identify genes that are altered during Cry11Aa intoxication, I identified an N-cadherin gene (AAEL000597) that was significantly down-regulated. An EGF-LamG fragment from this N-cadherin bound Cry11Aa with high affinity and competed with Cry11Aa binding to mosquito midgut membranes. Moreover, N-cadherin-silenced mosquitoes showed tolerance to Cry11Aa, implying that this cadherin is involved in mediating Cry11Aa toxicity. I also showed that this N-cadherin interacts with an amino acid on loop α-8 of Cry11Aa, which is different from that which interacts with the Aedes cadherin binding region. These data suggest that Cry11Aa probably has two different pathogenic pathways that act through two different cadherins in Ae. aegypti.

I also established a Cry11A-resistant strain to determine which mode of action is involved in Aedes Cry11Aa resistance. Brush border membranes from this strain (G30) bound Cry11Aa less compared to the binding in the wild type (WT), implying Cry11Aa resistance results from altered receptor binding affinity, but not proteolytic activity since no change in the latter was observed. Using RNA-seq analyses, immunoblot assays and mass spectrometry, we found the N-cadherin (AAEL000597) and an alkaline phosphatase (ALP, AAEL003298) were down-regulated in Cry11A-resistant larvae midgut. These results strongly suggest that N-cadherin and ALP are associated with Cry11Aa resistance in Ae. aegypti.

In summary, based on data in the literature and my work, I demonstrate that two different pathways of Cry11Aa toxicity are possible; one involving an N-cadherin and the other a combination of Aedes cadherin and an ALP in Ae. aegypti. Moreover, N-cadherin and ALP not only mediate Cry11Aa toxicity, but they were appear to be associated with Cry11Aa resistance. ALP has been proposed as a secondary receptor mediating Cry11Aa toxicity with Aedes cadherin, and hence attenuation of its expression can lead to Cry11A resistance.

    Cover page: Toxin Binding Receptors and the Mode of Action of Bacillus thuringiensis subsp. israelensis Cry Toxins
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2014)
    Bacillus thuringiensis subsp. israelensis (Bti) is widely used for the biological control of mosquito populations. However, the mechanism of Bti toxins is still not fully understood. To further elucidate the mechanism of Bti toxins, we developed an Aedes aegypti resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa (strain G30). G30 larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae (WT). Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to WT BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using Illumina sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the WT. None of the transcripts for previously identified receptors of Cry11Aa (Aedes cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to WT. However, ALP proteins were expressed at reduced levels (∼ 40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in Ae. aegypti.
      Cover page: Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2015)
      Cadherin plays an important role in the toxicity of Bacillus thuringiensis Cry proteins. We previously cloned a full-length cadherin from Aedes aegypti larvae and reported this protein binds Cry11Aa toxin from B. thuringiensis subsp. israelensis with high affinity, ≈16.7nM. Based on these results, we investigated if Aedes cadherin is involved in the in vivo toxicity of Cry11Aa toxin to Ae. aegypti. We established a mosquito cell line stably expressing the full-length Aedes cadherin and transgenic mosquitoes with silenced Aedes cadherin expression. Cells expressing the Aedes cadherin showed increased sensitivity to Cry11Aa toxin. Cry11Aa toxin at 400nM killed approximately 37% of the cells in 3h. Otherwise, transgenic mosquitoes with silenced Aedes cadherin expression showed increased tolerance to Cry11Aa toxin. Furthermore, cells expressing Aedes cadherin triggered Cry11Aa oligomerization. These results show the Aedes cadherin plays a pivotal role in Cry11Aa toxicity to Ae. aegypti larvae by mediating Cry11Aa oligomerization. However, since high toxicity was not obtained in cadherin-expressing cells, an additional receptor may be needed for manifestation of full toxicity. Moreover, cells expressing Aedes cadherin were sensitive to Cry4Aa and Cry11Ba, but not Cry4Ba. However transgenic mosquitoes with silenced Aedes cadherin expression showed no tolerance to Cry4Aa, Cry4Ba, and Cry11Ba toxins. These results suggest that while Aedes cadherin may mediate Cry4Aa and Cry11Ba toxicity, this cadherin but is not the main receptor of Cry4Aa, Cry4Ba and Cry11Ba toxin in Ae. aegypti.
        Cover page: Aedes cadherin mediates the in vivo toxicity of the Cry11Aa toxin to Aedes aegypti
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