Article

Bacillus thuringiensis Cry1Ab, but not Cry1Aa or Cry1Ac, disrupts liposomes

Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan; Advanced Technology Institute, Kubota Corporation, Ibaraki 301-0852, Japan; Graduate School, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
Pesticide Biochemistry and Physiology (Impact Factor: 2.11). 01/2006; DOI: 10.1016/j.pestbp.2005.02.001

ABSTRACT 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.

0 Bookmarks
 · 
101 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 microg Cry1Ac/g diet but killed by Cry1Ab at 0.5 microg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 microg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.
    Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 09/2007; 147(4):716-24. · 2.07 Impact Factor
  • Source
    [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。
  • Source
    [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).
    01/2007;