Peptidoglycan recognition protein S2 from silkworm integument: characterization, microbe-induced expression, and involvement in the immune-deficiency pathway

Abstract

Peptidoglycan recognition protein (PGRP) binds specifically to peptidoglycan and plays an important role as a pattern recognition receptor in the innate immunity of insects. The cDNA of a short-type PGRP, an open reading frame of 588 bp encoding a polypeptide of 196 amino acids, was cloned from Bombyx mori. A phylogenetic tree was constructed, and the results showed that BmPGRP-S2 was most similar to Drosophila melanogaster PGRP (DmPGRP-SA). The induced expression profile of BmPGRP-S2 in healthy Escherichia coli- and Bacillus subtilis-challenged B. mori was measured using semiquantitative reverse transcriptase polymerase chain reaction analysis. The expression of BmPGRP-S2 was upregulated at 24 h by E. coli and Ba. subtilis challenge. In addition, in the integument of B. mori, RNAi knockdown of BmPGRP-S2 caused an obvious reduction in the transcription expression of the transcription factor Relish and in antibacterial effector genes Attacin, Gloverin, and Moricin. The results indicated that BmPGRP-S2 participates in the signal transduction pathway of B. mori.

Keywords: Bombyx mori; RNA interference; innate immunity; peptidoglycan recognition protein.

Fig. 1.

Nucleotide and the deduced amino acid sequences of the encoding region of BmPGRP-S2. The signal peptide is underlined, the PGRP domain is gray, the Ami_2 domain is boxed, the star is the stop codon, the initiation codon is double underlined, and there is a different base between the results of cloning and the draft sequence of the Bombyx genome, which is indicated by a circle.

Fig. 2.

Multisequence alignment of BmPGRP-S1 (NP_001036836) and BmPGRP-S2 with BTL-LP2, B. mori mRNA for bacteriophage T7 lysozyme-like protein 2 (BAB33295). Amino acid residues that are conserved sequences are shaded dark, and similar amino acids are shaded gray. Zn²⁺ binding sites and amidase catalytic sites are marked with a circle and triangle, respectively.

Fig. 3.

Phylogenetic tree of BmPGRP-S2 with PGRPs from Drosophila. The tree is constructed by the neighbor-joining (NJ) algorithm using Mega 4.0 based on the multiple-sequence alignment by ClustalW. The numbers above the branch represent bootstrap percentages. The topology was tested using bootstrap analyses (500 replicates). The protein sequences used for phylogenetic analysis were as follows: DmPGRP-SA (AAF48056.1), DmPGRP-SB1 (CAD89138.1), DmPGRP-SB2 (CAD89150.1), DmPGRP-SC1A (CAD89163.1), DmPGRP-SC1B (CAD89174.1), DmPGRP-SC2 (CAD89187.1), DmPGRP-SD (CAD89 193.1), DmPGRP-LA (AGB94318.1), DmPGRP-LB (AFH06372.1), DmPGRP-LC (AFH04364.1), DmPGRP-LD (AAO41277.1), DmPGRP-LE (AAG32064.1), and BTL-LP2 (BAB33295.1).

Fig. 4.

Upregulation of BmPGRP-S2 mRNA as determined by RT-PCR 24 h after injection of Ba. subtilis and E. coli into B. mori larvae on day 3 of the fifth instar compared with the ddH2O-injected control. eTIF4A was used as an internal control gene.

Fig. 5.

Downregulation of Relish (B) and of the AMP genes Attacin, Gloverin, and Moricin (C) by RNAi knockdown of BmPGRP-S2 (A). Newly exuviated fifth-instar larvae were used for the experiment. The mRNA levels of the genes in the integument of silkworms were determined by RT-PCR. +dsRNA, BmPGRP-S2 ds-RNA injection; -dsRNA, no BmPGRP-S2 ds-RNA injection; eTIF4A was used as an internal control gene.