Detection of ß-exotoxin synthesis in Bacillus thuringiensis using an easy bioassay with the nematode Caenorhabditis elegans.

UNLABELLED: The insecticidal activity of Bacillus thuringiensis is owing to the action of Cry and Cyt proteins. In addition to the synthesis of insecticidal proteins, some strains are able to synthesize ß-exotoxin, which is highly toxic to humans. In this regard, it is very important to have a simple method to detect ß-exotoxin to avoid the commercial production of this type of strains. In this work, we developed a simple and fast method, using the nematode Caenorhabditis elegans to detect indirectly the synthesis of ß-exotoxin by B. thuringiensis strain. Using this assay, we detected that ~60% of Mexican native strains (i.e. LBIT-471, 491, 492, 497, 507, 511, 515, 536 and 537) were toxic to the nematode (44-97% mortalities) and their ß-exotoxin (ßEx(+) ) production, including a positive control (NRD-12), was confirmed by HPLC. In addition, the negative controls (ßEx(-) ) LBIT-436 (HD-1) and LBIT-438 and also the native strains LBIT-499, 500, 521, 522, 533 and 542, did not show a detrimental effect against nematodes larvae, neither the synthesis of ß-exotoxin as determined by HPLC. Finally, we did not find a correlation between B. thuringiensis strains with similar plasmid patterns and the ß-exotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we implemented a qualitative and fast bioassay using the nematode Caenorhabditis elegans to detect the production of ß-exotoxin in different strains of Bacillus thuringiensis. We show that this assay is useful to detect ß-exotoxin in B. thuringiensis with high reliability, helping to discriminate strains that could not be used as bioinsecticides because of their putative risk to humans. Data show that qualitative bioassay with nematodes is a potential alternative to fly larvae bioassays, and correlated with the determination of ß-exotoxin by HPLC.

Total synthesis and conformational studies of hapalosin, N-desmethylhapalosin and 8-deoxyhapalosin.

Hapalosin (2), a 12-membered cyclic depsipeptide possessing MDR-reversing activity, and analogues (3) and (4) have been synthesized using macrolactamization as an important ring-forming step. Three building blocks: (2S,3R)-3-(tert-butyldimethylsilyloxy)-2-methyl-decanoic acid (13), benzyl (S)-2-hydroxy-3-methylbutanate (14), and (4S,3R)-4-(benzyloxycarbonyl-methylamino)-3-methoxymethoxy-5-pheny l-pentanoic acid (28) were prepared from Evans's chiral imide (9), L-valine, and L-N-Boc phenylalanine (17), respectively, and were assembled together by applying twice Yamaguchi's coupling methodology. A new and efficient selective N-methylation of gamma-hydroxy-beta-amino ester taking advantage of the vicinal amino alcohol function was uncovered in the course of this study. Thus, treatment of compound 19 with HCHO in the presence of catalytic amount of pTsOH followed by reduction (NaBH3CN, TFA, CH2Cl2) of the so-formed oxazolidine 24 gave the N-methylated product 25. Furthermore, a dual role of oxazolidine as protecting group of vicinal amino alcohol and latent N-methyl function was established which allowed synthesizing both hapalosin (2) and N-desmethylhapalosin (3) from the same linear precursor 32 in a step-efficient and atom economic way. In contrast to hapalosin (2) and N-desmethyl analogue (3), the amide bond of 8-deoxy hapalosin (4) exists at room temperature (CDCl3) exclusively in s-cis conformation as evidenced by NOE studies. This observation has been explained on the basis of computational studies. No significant MDR reversing activity of 8-deoxy hapalosin (4) was observed in K562 R and S/Adriblastine against human erythroleucemic cell lines indicating thus the important contribution of hydroxy group to the bioactivity of hapalosin.