Role Of HaPGRP-D Gene In Innate Immunity Of The Cotton Bollworm, Helicoverpa Armigera

Peptidoglycan recognition proteins （PGRPs） specifically bind to peptidoglycans, and play crucial roles as pattern recognition receptors （PRRs） in mediating innate immune responses. In this study, we identified and characterized a PGRP （HaPGRP-D） from the cotton bollworm, Helicoverpa armigera. We preliminary investigated the functions of HaPGRP-D in H. armigera innate immunity. The results are as follows:1. Identification and sequence analysis of HaPGRP-D geneA PGRP cDNA was identified from the transcriptome of H. armigera hemocytes and named HaPGRP-D. The HaPGRP-D cDNA contained an open reading frame （ORF） of 699 bp. The predicted protein encoded by HaPGRP-D was 232-aa long and included a signal peptide （1-18 aa）, a PGRP domain （38-181 aa）, and an overlapping Ami₂ domain （50-187 aa）. The mature HaPGRP-D had a predicted molecular weight （MW） of 26.081 kDa and a theoretical isoelectric point （PI） of 5.82.Multiple sequence alignment of the deduced protein sequences of the lepidopteran PGRP genes indicated that HaPGRP-D amino acid sequence had significant similarities with the PGRP sequences from other lepidopterans. Comparative analysis indicated that 5 amino acids that were required for T7 lysozyme Zn2+binding and amidase activity were also conserved in HaPGRP-D, suggesting that HaPGRP-D is an amidase-type PGRP.2. Tissue distribution of HaPGRP-D mRNAsExpression of HaPGRP-D mRNAs was examined using RT-PCR in 4 tissues （hemocytes, fat body, midgut, and epidermis） of H. armigera larvae. HaPGRP-D was expressed at high levels in the midgut and low levels in the hemocytes. No expression was detected in the epidermis and fat body.3. Expression profiles of HaPGRP-D induced by bacterial or chromatography beads challengeqRT-PCR was used to measure the changes in transcript levels of HaPGRP-D in hemocytes of H. armigera larvae after the injection of bacteria or beads. Our results showed that the expression of HaPGRP-D in hemocytes of H. armigera was upregulated after injecting Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, or chromatography beads.4. Prokaryotic Expression and Purification of HaPGRP-DThe sequence encoding mature peptide of HaPGRP-D cDNA was amplified using a pair of gene specific primers. The DNA fragments were cloned into expression vector pET-32a. The recombinant plasmids pET-32a-PGRP-D were transformed into competent E. coli BL21 cells, and protein expression was induced by IPTG. Recombinant HaPGRP-D was purified using the High-Affinity Ni-NTA Resin.The molecular weight of rHaPGRP-D was about 40 kDa.5. HaPGRP-D protein activity analysis in vitroThe rHaPGRP-D was carried out the following protein activity analysis experiment in vitro:5.1 Binding activity to Bacteria analysisE. coli and S. aureus were used to test the microbial binding activity of rHaPGRP-D. The results indicated that HaPGRP-D could directly bind to bacteria in a zinc-dependent manner.5.2 Bacterial agglutination activities analysisThe agglutination activities of rHaPGRP-D toward E. coli and S. aureus were assessed.The results indicated that agglutination activities of HaPGRP-D toward bacteria were Zn2+-dependent.5.3 Amidase activity analysisInsoluble PGNs were purified from S. aureus and used to measure the amidase activity of rHaPGRP-D.The result indicating its high-degrading activity toward PGNs in the presence of Zn2+. Much lower degrading activity toward PGNs was detected in the non-Zn2+group. These results proved that HaPGRP-D is a Zn2+-dependent amidase.5.4 Antibacterial activity assayThe antibacterial activity of rHaPGRP-D was determined against E. coli and S. aureus.The results indicating that in the presence or absence of Zn2+, rHaPGRP-D showed an apparent growth inhibition of E. coli. However, only in the presence of Zn2+, rHaPGRP-D showed significant antibacterial activity against S. aureus.5.5 HaPGRP-D promotes hemocyte phagocytosis analysisAn in vitro phagocytosis assay was performed to test whether HaPGRP-D was involved in the phagocytosis response. Compared with rTrx, rHaPGRP-D significantly enhanced the phagocytic activity of hemocytes against bacteria both with respect to phagocytic rate （PR） and phagocytic index （PI）. These results indicated that HaPGRP-D may be involved in the phagocytosis process of H. armigera hemocytes.5.6 HaPGRP-D promotes hemocyte encapsulationAn in vitro encapsulation assay was performed to test whether HaPGRP-D was involved in encapsulation response. Encapsulation rate of the beads encapsulated by hemocytes in rHaPGRP-D experimental group was significantly higher than in control group. These results suggested that HaPGRP-D may be involved in the encapsulation response of H. armigera hemocytes.5.7 Binding of HaPGRP-D to hemocytesWe conducted an immunocytochemistry assay to test whether HaPGRP-D could bind to hemocytes of H. armigera larvae. The results showed that rHaPGRP-D could bind to the surface of plasmatocytes and granulocytes.Along those lines, these results suggest that HaPGRP-D plays important roles as PRR, amidase, and opsonin in H. armigera humoral and cellular immune responses.