Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

Mitochondrial dysfunction critically contributes to many major human diseases. The impact of specific gut microbial metabolites on mitochondrial functions of animals and the underlying mechanisms remain to be uncovered. Here, we report a profound role of bacterial peptidoglycan muropeptides in promoting mitochondrial functions in multiple mammalian models. Muropeptide addition to human intestinal epithelial cells (IECs) leads to increased oxidative respiration and ATP production and decreased oxidative stress. Strikingly, muropeptide treatment recovers mitochondrial structure and functions and inhibits several pathological phenotypes of fibroblast cells derived from patients with mitochondrial disease. In mice, muropeptides accumulate in mitochondria of IECs and promote small intestinal homeostasis and nutrient absorption by modulating energy metabolism. Muropeptides directly bind to ATP synthase, stabilize the complex, and promote its enzymatic activity in vitro, supporting the hypothesis that muropeptides promote mitochondria homeostasis at least in part by acting as ATP synthase agonists. This study reveals a potential treatment for human mitochondrial diseases.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the potential function of PGRPs in the giant triton snail Charonia tritonis. In this study, a short-type PGRP gene (termed Ct-PGRP-S1) was identified in C. tritonis. Ct-PGRP-S1 was predicted to contain several structural features known in PGRPs, including a typical PGRP domain (Amidase_2) and Src homology-3 (SH3) domain. The Ct-PGRP-S1 gene was constitutively expressed in all tissues examined except in proboscis, with the highest expression level observed in the liver. As a typical PRR, Ct-PGRP-S1 has an ability to degrade peptidoglycan (PGN) and was proven to have non-Zn2+-dependent amidase activity and antibacterial activity against Vibrioalginolyticus and Staphylococcus aureus. It is the first report to reveal the peptidoglycan recognition protein in C. tritonis, and these results suggest that peptidoglycan recognition protein Ct-PGRP-S1 is an important effector of C. tritonis that modulates bacterial infection resistance of V. alginolyticus and S. aureus, and this study may provide crucial basic data for the understanding of an innate immunity system of C. tritonis.

Tumor-induced host wasting and mortality are general phenomena across species. Many groups have previously demonstrated endocrinal impacts of malignant tumors on host wasting in rodents and Drosophila. Whether and how environmental factors and host immune response contribute to tumor-associated host wasting and survival, however, are largely unknown. Here, we report that flies bearing malignant yki3SA-gut tumors exhibited the exponential increase of commensal bacteria, which were mostly acquired from the environment, and systemic IMD-NF-κB activation due to suppression of a gut antibacterial amidase PGRP-SC2. Either gut microbial elimination or specific IMD-NF-κB blockade in the renal-like Malpighian tubules potently improved mortality of yki3SA-tumor-bearing flies in a manner independent of host wasting. We further indicate that renal IMD-NF-κB activation caused uric acid (UA) overload to reduce survival of tumor-bearing flies. Therefore, our results uncover a fundamental mechanism whereby gut commensal dysbiosis, renal immune activation, and UA imbalance potentiate tumor-associated host death.

Pharyngitis is an inflammation of the pharynx caused by viral, bacterial, or non-infectious factors. In the present study, the anti-inflammatory efficacy of carvacrol was assessed using an in vitro model of streptococcal pharyngitis using human tonsil epithelial cells (HTonEpiCs) induced with Streptococcus pyogenes cell wall antigens. HTonEpiCs were stimulated by a mixture of lipoteichoic acid (LTA) and peptidoglycan (PGN) for 4 h followed by exposure to carvacrol for 20 h. Following exposure, interleukin (IL)-6, IL-8, human beta defensin-2 (HBD-2), epithelial-derived neutrophil-activating protein-78 (ENA-78), granulocyte chemotactic protein-2 (GCP-2), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), and prostaglandin (PGE2) were measured by enzyme-linked immunosorbent assays (ELISA). The levels of pro-inflammatory cytokines, IL-6, IL-8, ENA-78, and GCP-2 were decreased in a carvacrol dose-dependent manner. The production of HBD-2 was significantly suppressed over 24 h carvacrol treatments. PGE2 and COX-2 levels in the cell suspensions were affected by carvacrol treatment. TNF-α was not detected. The cell viability of all the tested carvacrol concentrations was greater than 80%, with no morphological changes. The results suggest that carvacrol has anti-inflammatory properties, and carvacrol needs to be further assessed for potential clinical or healthcare applications to manage the pain associated with streptococcal pharyngitis.

Peptidoglycan (PGN) is a unique component in the cytoderm of prokaryotes which can be recognized by different pathogen-associated molecular patterns (PAMPs) in eukaryotes, followed by a cascade of immune responses via different pathways. This review outlined the basic structure of PGN, its immunologic functions. The immunomodulation pathways mediated by PGN were elaborated. PGN induces specific immunity through stimulating different cytokine release and Th1/Th2-dominated immune responses during humoral/cellular immune response. The nonspecific immunity activation by PGN involves immunomodulation by different pattern recognition receptors (PRRs) including PGN recognition proteins (PGRPs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), Toll-like receptors (TLRs), and C-type lectin receptors (CLRs). The sources and classification of PGRPs were summarized. In view of the stimulating activities of PGN and its monomers, the potential application of PGN as vaccine or adjuvant was prospected. This review provides systematic information on PGN functionalities from the point of immunoregulation, which might be useful in the deep exploitation of PGN.Key points. The immunological functions of PGN were illustrated. Cellular and humoral immunomodulation by PGN were outlined. The use of PGN as vaccine or adjuvant was prospected.

The symbiotic relationship between commensal microbes and host animals predicts unidentified beneficial impacts of individual bacterial metabolites on animal physiology. Peptidoglycan fragments (muropeptides) from the bacterial cell wall are known for their roles in pathogenicity and for inducing host immune responses. However, the potential beneficial usage of muropeptides from commensal bacteria by the host needs exploration. We identified a striking role for muropeptides in supporting mitochondrial homeostasis, development, and behaviors in Caenorhabditis elegans. We determined that the beneficial molecules are disaccharide muropeptides containing a short AA chain, and they enter intestinal-cell mitochondria to repress oxidative stress. Further analyses indicate that muropeptides execute this role by binding to and promoting the activity of ATP synthase. Therefore, given the exceptional structural conservation of ATP synthase, the role of muropeptides as a rare agonist of the ATP synthase presents a major conceptual modification regarding the impact of bacterial cell metabolites on animal physiology.

The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.

Peptidoglycan recognition proteins (PGRPs), which are discovered in invertebrates and vertebrates, play an important role in antibacterial immunity. However, the function of PGRPs is largely uninvestigated in reptiles. In the present study, a short-type PGRP gene, designed as C-turtle-PGRP-S, was identified in the Chinese soft-shelled turtle, Pelodiscus sinensis. The C-turtle-PGRP-S contains a highly conserved PGRP domain and has close relationship with PGRP-S orthologues in other species according to sequence and phylogenetic analyses. C-turtle-PGRP-S gene was constitutively expressed in all detected tissues and was induced by Edwardsiella tarda. Additionally, recombinant C-turtle-PGRP-S showed PGN binding activity and antibacterial function against E. tarda. Therefore, it is suggested that the function of PGRP-S is likely to be conserved in reptile vertebrates, as observed in other vertebrates, shedding light on the evolutionary conservation of PGRPs.

Antimicrobial peptide (AMP) is a crucial component of the innate immune system in crustaceans. In mud crab, Scylla paramamosain, a commercially important species, a glycine-rich antimicrobial peptide (Spgly-AMP) gene was newly identified and putatively encoded a 26aa signal peptide and 37aa mature peptide. To understand the function of Spgly-AMP, the expression profile of Spgly-amp gene was characterized, which showed Spgly-amp was expressed widely in most tissues of adult crabs with the highest expression level in hemocytes. After Vibrio parahaemolyticus, PGN, or Poly I:C stimulations, the expression level of Spgly-amp was significantly up-regulated in the hemocytes. In antimicrobial assays, chemically synthesized Spgly-AMP peptides exhibited strong antibacterial activities against both Gram-positive and Gram-negative bacteria and high thermal stability after high-temperature heating. These findings in the present study verified the importance of the Spgly-AMP in defense of pathogenic bacteria infection in the mud crab and provided a promising candidate of antimicrobial agents in the crab aquaculture.