Molecular cloning and characterization of a short peptidoglycan recognition protein (PGRP-S) with antibacterial activity from the bumblebee Bombus ignitus

Abstract

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that recognize peptidoglycan, a unique bacterial cell wall component. Here we cloned and characterized PGRP-S from the bumblebee Bombus ignitus ( BiPGRP-S). The BiPGRP-S gene consists of four exons that encode 194 amino acid residues. Comparative analysis indicates that the predicted amino acid sequence of BiPGRP-S shares a high identity with enzymatically active PGRP-S proteins and contains the amino acids required for amidase activity. BiPGRP-S in B. ignitus worker bees is constitutively expressed in both the fat body and epidermis, and it is secreted into the hemolymph. Quantitative real-time PCR assays revealed that the BiPGRP-S gene is highly induced in both the fat body and the epidermis after an injection of Bacillus thuringiensis. In addition, recombinant BiPGRP-S expressed as a 19-kDa protein in baculovirus-infected insect cells can bind to Bacillus megaterium and B. thuringiensis but not to Staphylococcus aureus, Escherichia coli or Beauveria bassiana. Consistent with these data, BiPGRP-S shows antibacterial activity against B. megaterium and B. thuringiensis. After B. thuringiensis injection, the expression profiles of four antibacterial peptide genes in the fat body of RNA interference (RNAi)-mediated BiPGRP-S-knock-down B. ignitus worker bees was similar to that of control worker bees, indicating that BiPGRP-S does not affect the activation of antibacterial peptide gene expression. These results indicate that BiPGRP-S is an inducible protein that may function as an amidase-type PGRP-S during the immune response against Bacillus bacteria.