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The Cytotoxicity of Binary Toxin PirAB from Photorhabdus Luminescence

Author: LiYuZuo
Tutor: HuShengBiao
School: Hunan Normal University
Course: Microbiology
Keywords: binary toxin cytotoxicity injectable toxicity
CLC: S482.39
Type: Master's thesis
Year: 2014
Downloads: 3
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Photorhabdus luminescens are gram-negative, insect pathogenic bacteria which forming mutualism with Heterorhabditis and colonizing in the gut of nematodes. The bacteria multiply in the hemolymph of larval-stage insects, releasing plenty of toxins to kill target larval. The main four toxins contain Tc toxin complexes, Mcf, PVC and PirAB, most of them exhibit cytotoxicity. Tc toxin complexes exploit syringe-like injection mechanism to deliver toxin into target cells. Mcf rearranges cytoskeleton and induces cells apoptosis, leading to caterpillar floopy. PVC destroys insect hemocytes which undergo cytoskeleton condensation. By far, research concerning cytotoxicity of PirAB is rare. However, PirAB not only presents oral and injectable toxicity to several agricultural pests such as wax moth and diamondback moth, it also shows activity against Anopheles gambiae, Culex pipiens and Aedes aegypti. In order to elucidate the cytotoxicity of PirAB and to explore its role to insect humoral immunity, the PirAB was heterologously expressedand its cytotoxicty was studied.In this study, we cloned plu4092-plu4093which encoded PirAB from P. luminescens TT01genome and expression vector pETDuet-pirAB were constructed, then we transferredit into E.coli Rosetta (DE3). After induction with IPTG, the recombinant protein showed great solubility. We purified recombinant protein by Ni-NTA and desalted by dialysis. When incubated PirAB with midgut cell lines CF-203from Choristoneura fumiferana, MTT-bioassay shows toxicity of PirAB to cells largely depends on its concentration. After24h incubation, the morphology of cells no longer remained round, the membrane edge wrinkled and contents leaking was observed. PirAB affected membrane permeability through trypan blue staining. The confocal laser microscopy showed fluorescence intensity of tubulin greatly weakened compared to control. We speculated that tubulin network depolymerized under the influence of PirAB, the potent target of PirAB may be microtubulin. The extracted DNA of PirAB treated-cells showed fragmentation, the cells may undergo apoptosis.We also constructed PirAB fusion toxin and studied its toxicity. Through a flexible linker, plu4092linked to plu4093and it was under regulation of promoter and terminator of crylAc. The expression vector pHT1AcPT-fusion was transferred into Bacillus thuringiensis crystal-minus XBU001which efficiently expressingPirAB formed inclusion body. After fermentation,purification and dissolution, the fusion protein triggered melanism and mortality of fourth instar Spodoptera exiguaby injection.This study elucidated cytotoxity and the potential pathogenic mechanism of PirAB, laying a foundation of PirAB in P. luminescens to insect cellular immunity. The injectable toxicty of PirAB against fourth instar Spodoptera exigua presented a potent development of new insecticides.

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