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The bacterium  Photorhabdus luminescens  produces a number of insecticidal proteins to kill its larval prey. In this study, we cloned the gene coding for a binary toxin PirA 1 B 1  and purified the recombinant protein using affinity chromatography combined with desalination technology. Furthermore, the cytotoxicity of the recombinant protein against the haemocytes of  Galleria mellonella  larvae was investigated. We found that the protein had haemocoel insecticidal activity against  G. mellonella  with an LD50 of 131.5 ng/larva. Intrahaemocoelic injection of PirA 1 B 1  into  G. mellonella  resulted in significant decreases in haemocyte number and phagocytic ability. In  in vitro  experiments, PirA 1 B 1  inhibited the spreading behaviour of the haemocytes of  G. mellonella  larvae and even caused haemocyte degeneration. Fluorescence microscope analysis and visualization of haemocyte F-actin stained with phalloidin-FITC showed that the PirA 1 B 1  toxin disrupted the organization of the haemocyte cytoskeleton. Our results demonstrated that the PirA 1 B 1  toxin disarmed the insect cellular immune system.

Haemocoel injection of PirA1B1 to Galleria mellonella larvae leads to disruption of the haemocyte immune functions