We evaluated the ability of Cry1Aa9, Cry1Ab4, and Cry1Ac1 insecticidal toxins from Bacillus thuringiensis to destroy liposomes. Cry1A toxins are thought to form pores in midgut apical cell membranes (BBMV), thereby disrupting midgut cells. Liposomes containing fluorescent calcein were prepared using phosphatidylcholine (PC) and phosphatidylserine (PS) (PC/PS-Liposomes) or PC alone (PC-Liposomes). Cry1Ab (1.4 μM), but not Cry1Aa or Cry1Ac, disrupted PC/PS-Liposomes and PC-Liposomes. PC/PS-Liposomes containing cholesterol and oligosaccharylceramide from Plutella xylostella midgut were damaged even more extensively by Cry1Ab, but the inclusion of either lipid alone had no effect. The initial velocity of Cry1Ab-mediated liposome disruption increased 17-fold when liposomes were prepared with Triton X-100-soluble proteins from Bombyx mori BBMV and PC (PC/Proteo-Liposomes), and Cry1Aa and Cry1Ac also caused slight disruption. These data suggest that Cry1Ab achieves higher penetration into PC/PS-Liposomes, PC-Liposomes, and PC/Proteo-Liposomes compared with Cry1Aa or Cry1Ac and that Cry1Ab may interact with membrane proteins.
"Cry1Aa and Cry1Ab have a high degree of sequence similarities within domain III; on the other hand, Cry1Ab and Cry1Ac have high similarities within domain II. Therefore, only the structure of the border between domains II and III differs among these three proteins (Kato et al. 2006). The Crickmore database collects all the cited sequences of each Cry protein (http://www.lifesci.sussex.ac.uk/home/ neil_Crickmore/Bt/index.html. "
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.
Archives of Microbiology 04/2014; 196(6). DOI:10.1007/s00203-014-0981-3 · 1.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Liposomes were constructed with phosphatidylcholine (PC) and fluorescence indicator, calcein, and purified using 9-40% discontinuous sucrose density gradients centrifugation (SDGC) or gel filtration column chromatography (GFCC). Stability and sensitivity of those liposomes to insecticidal Cry1A toxins of Bacillus thuringiensis were compared each other and one purified by GFCC was shown to leak calcein by 40% of total fluorescence contents during 96 hr storage. On the other hand, liposome purified by SDGC was more stable and almost no leakage observed during the same period. Cry1Ab of B. thuringiensis weakly reacted with the liposome purified by GFCC and 2.5% of total encapsulated fluorescence was released, but Cry1Aa and Cry1Ac did not. Contrarily, liposome purified by SDGC was highly reactive with Cry1Ab and 40% of the calcein was released and 15%-release by Cry1Aa and Cry1Ac were also observed. These data clearly indicated that liposomes purified by SDGC were more stable and highly reactive to Cry toxins compared to that purified by GFCC. These liposomes are useful tool to analyze the interaction between Cry1A toxins and lipid membranes.ホスファチジルコリン(PC)を用いてリポソームを構築し、内部空間に蛍光色素、カルセインを封入した。PCリポソームをショ糖密度勾配遠心法(SDGC)あるいはゲルろ過カラムクロマトラグラフィー(GFCC)で精製し、各々の精製PCリポソームについて、溶液中での安定性とBacillus thuringiensisのCry1A殺虫毒素との反応性を比較解析した。GFCCで精製したPCリポソームは不安定で、96時間保存すると内包カルセインの40%がリポソームの外に漏出した。一方、SDGC精製PCリポソームは安定で96時間保存後もほとんどカルセインの漏出を示さなかった。殺虫毒素Cry1AbはGFCC精製PCリポソーム膜に小孔形成を示したが、カルセインの漏出は2.5%にとどまり、小孔形成率は非常に低いと思われた。またCry1AaとCry1Acは共に小孔を形成しなかった。一方、SDGC精製PCリポソームはCry1Abと反応し、40%のカルセイン漏出が検出された。Cry1AaとCry1Acとの反応に際しても15%のカルセイン漏出が検出された。これらの実験結果は、SDGC精製PCリポソームの方がGFCC精製PCリポソームよりも安定で、Cry1A毒素との反応性も高いことを示し、比重に基づきリポソームを精製することが均一な安定リポソームを得るうえで有効であることを示した。SDGC精製PCリポソームはCry1A毒素と脂質膜との相互作用を解析する上で有効である。
[Show abstract][Hide abstract] ABSTRACT: The insecticidal mechanism of Cry toxins, especially the interaction between Cry toxins and larval midgut apical cell membranes (brush border membrane, BBM), has not been fully elucidated. We employed liposomes as a simplified model to study the interaction of Cry toxins with membranes and evaluate their destructive efficacy. Cry1Ab disrupted phosphatidylcholine/phosphatidylserine (PC/PS) liposomes and PC/PS liposomes containing cholesterol and oligosaccharylceramide from Plutella xylostella midgut more extensively. When PC liposomes containing Triton X-100 soluble proteins from BBM vesicles of Bombyx mori were exposed to Cry1Ab, the pore formation activity increased 17-fold compared to that in PC liposomes, suggesting that Cry1Ab achieved higher penetration into various liposomes than the two other toxins that were tested (Cry1Aa and Cry1Ac).
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