Cloning and expression analysis of a long type peptidoglycan recognition protein(PGRP-L) from Xenopus tropicalis

Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned in the lower vertebrate species Xenopus tropicalis（Xt）.The XtPGRP-L possessed a conserved genomic structure with five exons and four introns.The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP.The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb.During embryonic development,XtPGRP-L was not expressed until the 72 h tadpole stage.In adult tissues,it was strongly expressed in the liver,lung,intestine,and stomach.Furthermore,after LPS stimulation,the expression of XtPGRP-L was up-regulated significantly in the liver,intestine and spleen,indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.

Identification,Phylogeny and Expressional Profiles of Peptidoglycan Recognition Protein(PGRP)Gene Family in Sinonovacula constricta

Peptidoglycan recognition protein(PGRP)plays a vital role in invertebrate innate immunity system as a specific pattern recognition receptor for peptidoglycan.Bivalves possess various PGRP systems for self-defense;however,it has not been characterized in razor clam Sinonovacula constricta.In this study,eight PGRP coding sequences were identified and analyzed from S.constricta genome,which are designated as ScPGRP-S1,ScPGRP-S2,ScPGRP-S3,ScPGRP-S4,ScPGRP-S5,ScPGRP-S6,ScPGRP-S7,ScPGRP-S8.The results of molecular evolutionary analyses showed that all eight ScPGRP genes were highly conserved and exhi-bited a typical PGRP/amidase_2 domain as PGRP genes in other mollusks.Moreover,the presence of signal peptides was predicted in ScPGRP-S2,ScPGRP-S3 and ScPRP-S6,while a transmembrane structure only existed in ScPGRP-S6.Notably,a tertiary struc-ture analysis indicated that no disulfide bond was observed in ScPGRP-S5 and ScPGRP-S7.The mRNA transcripts analysis of ScPGRPs revealed that the high expression patterns of ScPGRP-S1 and ScPGRP-S4 were found in mantle,adductor muscle and foot,while those of ScPGRP-S2,ScPGRP-S3 and ScPGRP-S6 were observed in hepatopancreas.Furthermore,the temporal expression profiles of ScPGRPs in the hepatopancreas were analyzed by qPCR<https://www.sciencedirect.com/topics/immunology-and-microbiology/real-time-polymerase-chain-reaction>after Gram-negative Vibrio parahaemolyticus and Gram-positive Staphylococcus aureus challenges.The mRNA expressions of ScPGRP-S2,ScPGRP-S3 and ScPGRP-S6 could be induced by V.pa-rahaemolyticus and S.aureus.Overall,our findings indicated that ScPGRPs were involved in the immune defense against invaders,which constituted a comprehensive understanding of the potential role of PGRP genes in S.constricta.

Antiviral function of peptidoglycan recognition protein in Spodoptera exigua(Lepidoptera:Noctuidae)

Peptidoglycan recognition proteins(PGRPs)are a class of molecules that play a critical role in insect immunity.Understanding the function of PGRPs is important to improve the efficiency of microbial insecticides.In this study,we investigated the role of PGRP-LB(a long type PGRP)in insect immunity against viruses using Spodoptera exigua and Spodoptera exigua multiple nucleopolyhedrovirus(SeMNPV)as an insect-virus model.We cloned and identified a PGRP-LB gene from S.exigua;the gene consisted of 7 exons that encoded a polypeptide of 234 amino acids with a signal peptide and a typical amidase domain.Expression analysis revealed that the abundance of SePGRP-LB transcripts in the fat body was greater than in other tissues.Overexpression of SePGRP-LB resulted in a significant decrease of 49%in the rate of SeMNPV-infected cells.In addition,the multiplication of SeMNPV was significantly decreased:a decrease of 79%in the production of occlusion-derived virion(ODV),and a maximum decrease of 50%in the production of budded virion(BV).In contrast,silencing of SePGRP-LB expression by RNA interference resulted in a significant 1.65-fold increase in the rate of SeMNPV-infected cells,a significant 0.54-fold increase in ODV production,a maximum 1.57-fold increase in BV production,and the larval survival dropped to 21%.Our findings show that SePGRP-LB has an antiviral function against SeMNPV,and therefore this gene may provide a target for lepidopteran pest control using virus insecticides.

Comparative analysis of peptidoglycan recognition proteins in endoparasitoid wasp Microplitis mediator

Peptidoglycan recognition proteins （PGRPs） are a family of innate immune receptors that specifically recognize peptidoglycans （PGNs） on the surface of a number of pathogens. Here, we have identified and characterized six PGRPs from endoparasitoid wasp, Microplitis mediator （MmePGRPs）. To understand the roles of PGRPs in parasitoid wasps, we analyzed their evolutionary relationship and orthology, expression profiles during different developmental stages, and transcriptional expression following infection with Gram-positive and -negative bacteria and a fungus. MmePGRP-S1 was significantly induced in response to pathogenic infection. This prompted us to evaluate the effects of RNA interference mediated gene specific knockdown ofMmePGRP-S1. The knockdown of MmePGRP-S1 （iMmePGRP-S1） dramatically affected wasps＇ survival following challenge by Micrococcus luteus, indicating the involvement of this particular PGRP in immune responses against Gram-positive bacteria. This action is likely to be mediated by the Toll pathway, but the mechanism remains to be determined. MmePGRP-S 1 does not play a significant role in anti-fungal immunity as indicated by the survival rate of iMmePGRP-S wasps. This study provides a comprehensive characterization of PGRPs in the economically important hymenopteran species M. mediator.

A splice variant of PGRP-LC required for expression of antimicrobial peptides in Anopheles gambiae

Members of the peptidoglycan recognition protein （PGRP） family play essential roles in different manifestations of immune responses in insects. PGRP-LC, one of seven members of this family in the malaria vector Anopheles gambiae produced several spliced variants. Here we show that PGRP-LC, and not other members of the PGRP family nor the six members of the Gram-negative binding protein families, is required for the expression of antimicrobial peptide genes （such as CEC1 and GAM1） under the control of the Imd-Rel2 pathway in an A. gambiae cell line, 4a3A. PGRP-LC produces many splice variants that can be classified into three sub-groups （LC1, LC2 and LC3）, based on the carboxyl terminal sequences. RNA interference against one LC1 sub-group resulted in dramatic reduction of CEC1 and GAM1. Over-expression of LCla and to a lesser extent LC3a （a member of the LC1 and LC3 sub-group, respectively） in the 4a3A cell line enhances the expression of CEC1 and GAM1. These results demonstrate that the LC1-subgroup splice variants are essential for the expression of CEC1 and GAM1 in A. gambiae cell line.

Functional characterization of PGRP-LC1 of Anopheles gambiae through deletion and RNA interference

Peptidoglycan recognition proteins （PGRP） play an important role in innate immunity in insects through the activation of the Imd pathway, which has been shown to be required in the antibacterial response in insects and in the limitation of the number of Plasmodium berghei oocysts developing in mosquito midgut. The LCI gene of the PRGP family in Anopheles gambiae produces many products through alternative splicing. In this work, we demonstrate that PGRP-LC1a alone is sufficient to activate the Imd pathway in the A. gambiae L3-5 cell line through a combination of terminal or internal deletions, and RNA interference against endogenous PGRP-LC products. In the absence of endogenous PGRP-LC proteins, the integrity of the cytoplasmic domain is necessary for LCla function, while that of the extracellular domain is not. Moreover, the shorter the extracellular domain, the higher the activity for LC1 a. However, the removal of either the cytoplasmic or the extracellular PGRP-binding domain has little impact on the activity of LC 1 a in the presence of endogenous PGRP-LC proteins.