Peptidoglycan recognition protein-S5 functions as a negative regulator of the antimicrobial peptide pathway in the silkworm, Bombyx mori

Highlights

• Amidase activity of PGRP-S5 is necessary for activation of the proPO pathway.

• PGRP-S5 negatively regulates antimicrobial peptide generation in silkworms.

• PGRP-S5 acts as a sensor, modulator, and effector in silkworm humoral immunity.

Abstract

Prophenoloxidase (proPO), immune deficiency (IMD), and Toll are the major signaling pathways leading to melanization and antimicrobial peptide production in insect hemolymph. Peptidoglycan recognition proteins (PGRPs) act as receptors and negative regulators in these pathways, and some PGRPs exhibit antimicrobial activity. Previously, we demonstrated that silkworm PGRP-S5 recognizes peptidoglycans (PGs) and triggers activation of the proPO pathway. It also acts as a bactericide, via its amidase activity (Chen et al., 2014). Here, we generated a C177S site-mutated silkworm PGRP-S5 protein that lacked amidase activity but retained its PG-binding capacity. Functional studies showed that the mutation caused loss of its receptor function for activation of the proPO pathway, suggesting that processing of PG by PGRP-S5 is necessary for formation of the pathway initiation complex. Further, we found that PGRP-S5 negatively regulates antimicrobial peptides generation in an amidase-dependent manner, likely through the IMD pathway. Thus, silkworm PGRP-S5 acts as a sensor, a modulator, and an effector in the silkworm humoral immune system.

Introduction

Peptidoglycan (PG) recognition proteins (PGRPs) are characterized by a PGRP domain that is homologous to bacteriophage and bacterial type 2 amidase (Kang et al., 1998, Dziarski and Gupta, 2006), which hydrolyzes the bond between the N-acetylmuramyl group in the glycan strand and the l-alanine in the stem peptide of peptidoglycan. Some PGRPs possess amidase activity, but some PGRPs lost amidase activity during the course of their evolution (Mellroth et al., 2003, Dziarski and Gupta, 2006, Kurata, 2014). PGRPs act as sensors, regulators, and effectors in immune systems (Royet et al., 2011, Kurata, 2014). A recent study revealed that Drosophila melanogaster PGRP-LC, which primarily functions as a signal-transducing receptor in the immune deficiency (IMD) pathway (Choe et al., 2002, Gottar et al., 2002, Ramet et al., 2002), is required for robust presynaptic homeostatic plasticity in the fly nervous system (Harris et al., 2015).

As signal-transducing receptors, PGRPs specifically recognize bacterial PGs and trigger host immune responses, such as antimicrobial peptide (AMP) generation and phenoloxidase (PO)-catalyzed melanization. Charroux et al. (2009) and Kurata (2014) summarized the scenario of detection of bacteria by Drosophila PGRPs. PGRP-SA, SD, and Gram-negative binding protein-1 (GNBP-1) cooperate to detect extracellular Lys-type PG from bacterial cell walls and initiate activation of the Toll pathway; PGRP-LC or PGRP-LE detects extracellular meso-diaminopimelic acid (DAP)-type PG from the cell walls of most gram-negative bacteria and some gram-positive bacteria and activates the IMD pathway. Activation of the Toll and IMD pathways leads to transcription of AMP genes. PGRP-LE also mediates the proPO cascade upstream of the proPO-activating enzyme (Takehana et al., 2002, Takehana et al., 2004). Intracellular PGPR-LE recognizes DAP-type PG and activates autophagy to eliminate intracellular bacteria such as Listeria monocytogenes (Charroux et al., 2009, Yano et al., 2008). PGRPs have also been found to act as receptors in immune pathways in organisms other than Drosophila. For instance, Bombyx mori PGRPs (Yoshida et al., 1996, Chen et al., 2014), Tenebrio molitor PGRP-SA (Park et al., 2006), Holotrichia diomphalia PGRP-1 (Lee et al., 2004), Manduca sexta PGRP-1 (Sumathipala and Jiang, 2010), and Helicoverpa armigera PGRP-A (Li et al., 2015) have been shown to participate in the proPO activation cascade. Mainly based on RNA interference (RNAi) results, it has been suggested that PGRPs from species other than Drosophila are responsible for transcriptional induction of AMP genes after bacterial challenge. RNAi against PGRP-LC reduced transcription levels of AMP CEC1 and DEF1 in Anopheles gambiae at the early stage of infection by Staphylococcus aureus (Meister et al., 2009). RNAi against PGRP-LD in the mosquito Armigeres subalbatus decreased transcription levels of certain AMPs (Wang and Beerntsen, 2015). In the mosquito Aedes aegypti, PGRP-LC is a critical receptor that mediates antibacterial responses to both Escherichia coli and Micrococcus luteus (Wang and Beerntsen, 2015). In the red flour beetle Tribolium castaneum, PGRP-LA plays a major role as an IMD pathway sensor that responds to both gram-positive and gram-negative bacterial infection; PGRP-LC and PGRP-LE might also function as sensors in the IMD pathway to defend against gram-negative bacteria (Koyama et al., 2015). RNAi against PGRP-S1 in the endoparasitoid wasp Microplitis mediator down-regulated defensin-1 at the transcriptional level (Wang et al., in press).

Some PGRPs function as immune regulators. In Drosophila, PGRP-LA is a positive regulator of the IMD pathway in barrier epithelia (Gendrin et al., 2013). PGRP-LF acts as a specific negative regulator of the IMD pathway on the basis of competition with PGRP-LCa for binding to PGRP-LCx (Maillet et al., 2008, Basbous et al., 2011). Several PGRPs with proven or predicted amidase activity play roles as negative regulators in immune systems. In Drosophila, PGRP-LB mainly functions as a scavenger to break down PG and dampen the IMD pathway and consequently controls the bacterial load in the gut; PGRP-SC2 degrades PG in the hemolymph and controls systemic immune responses (Zaidman-Remy et al., 2006, Costechareyre et al., 2016). In tsetse flies, PGRP-LB scavenges PG and prevents IMD pathway activation, facilitating establishment of the symbiont Wigglesworthia glossinidia in the host (Wang and Aksoy, 2012). Drosophila PGRP-SC2 also functions as a longevity-promoting factor by preventing commensal dysbiosis, stem cell hyperproliferation, and epithelial dysplasia through down-regulation of the IMD pathway (Guo et al., 2014). In the housefly Musca domestica, RNAi against PGRP-SC caused over-expression of AMP genes and delayed pupation (Gao et al., 2015).

Some PGRPs exhibit amidase-dependent bactericidal activity. Drosophila PGRP-SB1 shows enzymatic activity towards DAP-type polymeric PG (Zaidman-Remy et al., 2011), and in vitro assays showed that it can kill Bacillus megaterium (Mellroth and Steiner, 2006). However, in vivo tests showed no protective effect after bacterial infection (Zaidman-Remy et al., 2011). Our previous study of silkworm PGRP-S5 also demonstrated that it can break down a broad range of PGs and kill both gram-positive and gram-negative bacteria (Chen et al., 2014). Zebrafish PGRPs possess both amidase and bactericidal activities (Li et al., 2007).

We have already shown that silkworm PGRP-S5 plays roles both as effectors that kill bacteria and as receptors for activation of the proPO pathway; we also hypothesize that it might play roles as an immune modulator (Chen et al., 2014). Here we present evidences supporting its function as a negative regulator to control AMP production through an amidase-dependent mechanism.