Preserved antibacterial activity of ribosomal protein S15 during evolution.

Conventional role of ribosomal proteins is ribosome assembly and protein translation, but some ribosomal proteins also show antimicrobial peptide (AMP) activity, though their mode of action remains ill-defined. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933-55, Aquificae RPS1933-55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial surface via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67 were toxic to mammalian cells, making them promising lead molecules for the design of novel AMPs against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.

Identification and functional characterization of a novel member of low-density lipoprotein receptor-related protein (LRP)-like family in amphioxus.

Low-density lipoprotein receptor-related protein (LRP) is a group of important endocytic receptors contributing to binding ligands and maintaining internal environment. In this study, we identified a soluble LRP-like molecule in the amphioxus B. japonicum, BjLRP, with an uncharacterized domain structure combination of LY-EGF-CRD-EGF-CRD. It was mainly expressed in the gill, muscle, notochord and testis, and was significantly up-regulated following the challenge with bacteria. Recombinant BjLRP was capable of interacting with both Gram-negative and positive bacteria as well as PAMPs including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Interestingly, recombinant LY peptide was also able to bind to the Gram-negative and positive bacteria as well as the PAMPs LPS, LTA and PGN. By contrast, none of recombinant EGF1, EGF2, CRD1 and CRD2 had affinity to the bacteria and the PAMPs. In addition, BjLRPΔLY had no affinity to the PAMPs, although BjLRPΔLY showed slight affinity to the bacteria. These suggest that the interaction of BjLRP with the bacteria and PAMPs was primarily attributable to the LY domain. It is clear that BjLRP is a novel pattern recognition protein capable of identifying and interacting with invading bacteria in amphioxus.

Alpha 2 macroglobulin is a maternally-derived immune factor in amphioxus embryos: New evidence for defense roles of maternal immune components in invertebrate chordate.

In fish, a series of maternal derived immune components have been identified in their eggs or embryos at very early stages, which are proposed to provide protections to themselves against pathogenic attacks from hostile environment. The phenomenon of maternal immunity has been also recorded in several invertebrate species, however, so far, very limited information about the maternal immune molecules are available. In this study, it was demonstrated maternal alpha2 macroglobulin (A2m) protein, an important innate immune factor, exists in the fertilized eggs of amphioxus Branchiostoma japonicum, an invertebrate chordate. Maternal mRNA of A2m was also detected in amphioxus embryos at very early developing stages. In addition, it was recorded that the egg lysate prepared from the newly fertilized eggs can inhibit the growth of both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus in a concentration dependent manner. The bacteriostatic activity can be reduced notably after precipitated A2m with anti-A2m antibody. Thus maternal A2m is partly attributed to the bacteriostatic activity. It was further demonstrated that recombinant A2m can bind to E. coli cells directly. All these points come to a result that A2m is a maternal immune factor existing in eggs of invertebrate chordate, which may be involved in defense their embryos against harmful microbes' attacks.

Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.