PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella

- In this paper, we cloned and heterologously expressed of pirA and pirB genes from X. nematophila HB310 and tested the recombinant proteins against selected lepidopteran pests.
- The PirA/B mixture and PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD50 of 2.718 μg/larva and 1.566 μg/larva, respectively.
- PirA/PirB protein mixture or PirAB was found to cause hemolymph cell shrinkage.
- Our results suggest that both PirA and PirB together were necessary to cause typical hemocoel insecticidal activity against G. mellonella.

PirAB (Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens. The purpose of this experiment is to identify the relevant information for this toxin gene in X. nematophila. The pirA, pirB and pirAB genes of X. nematophila HB310 were cloned and expressed in Escherichia coli BL21 (DE3) using the pET-28a vector. A PirAB-fusion protein (PirAB-F) was constructed by linking the pirA and pirB genes with the flexible linker (Gly)4 DNA encoding sequence and then efficiently expressed in E. coli. The hemocoel and oral insecticidal activities of the recombinant proteins were analyzed against the larvae of Galleria mellonella. The results show that PirA/B alone, PirA/B mixture, co-expressed PirAB protein, and PirAB-F all had no oral insecticidal activity against the second-instar larvae of G. mellonella. Only PirA/B mixture and co-expressed PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD50 of 2.718 μg/larva or 1.566 μg/larva, respectively. Therefore, we confirmed that PirAB protein of X. nematophila HB310 is a binary insecticidal toxin. The successful expression and purification of PirAB laid a foundation for further studies on the function, insecticidal mechanism and expression regulation of the binary toxin.

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