The Open Toxinology Journal vol:3 issue:special issue #002 pages:141-150
Starved first instar Aedes aegypti larvae were 35-fold more sensitive to Bacillus thuringiensis subsp. israelensis
(Bti) toxins than fed larvae. Feeding larvae Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with Escherichia coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) indicate that TMOF and Cry toxins are synergisitic. tmfA was cloned and expressed in the cyanobacterium Anabaena PCC 7120 and the hormone was purified by HPLC and identified by ELISA. The amount of TMOF synthesized by Anabaena was low (0.5 - 1 μg in 108 cells). P. pastoris, which synthesizes high amounts of heterologous proteins in the presence of methanol and is readily consumed by mosquito larvae, was genetically engineered to produce more TMOF. Codon-optimized synthetic genes, cry11Aa- tmfA and gst-cry11Aa- tmfA, that were cloned into P. pastoris and fed to Ae. aegypti larvae caused 87.5% mortality in 5 days. GST (glutathione-S-transferase)enhanced the activity of Cry11A-TMOF and protected it from heat denaturation. Cell free extracts of recombinant P.pastoris cells killed 40% of tested 4th instar larvae within 24 h, and mass spectra analysis confirmed that the recombinants synthesize Cry11Aa. This report shows for the first time that Cry toxins and TMOF are synergists to Ae. aegypti larvae when jointly fed or expressed in recombinant P. pastoris.