| Abstract: | Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.Human gp340, also known as DMBT1 (deleted in malignant brain tumors 1), belongs to the innate immune protein family of scavenger receptor cysteine-rich (SRCR)2 proteins, all of which contain one or more evolutionarily conserved SRCR domain linked to other conserved protein domains (1, 2). Many of these proteins serve as pattern recognition receptors for innate immunity. gp340 is expressed by epithelial cells and cells of the immune system, and its expression is up-regulated after inflammatory stimuli (3, 4). It inhibits bacterial invasion to epithelial cells and the secretion of proinflammatory cytokines (5–7). Thus, it appears to be an important mediator of host immune responses to various microbes and was recently linked to Crohn disease, a human inflammatory bowel disease (8). gp340 is also found in human secretions like tears and saliva, and the salivary form has long been known as salivary agglutinin, which is an important molecule in oral biofilm formation and is suggested to have a role in dental caries development (9–12). The mechanisms of gp340 action in these different biological contexts are not known.Common to all scavenger receptors, the ligand repertoire of gp340 is wide; it binds many different types of bacteria as well as viruses (10, 13, 14). The wide ligand recognition pattern of scavenger receptors is thought to be based on the recognition of common microbial structures, such as lipopolysaccharides and lipoteichoic acids, but in the case of gp340, specific bacterial surface proteins are reported to be involved in the interactions characterized (15–18). Because the importance of gp340/salivary agglutinin in the oral environment has been evident for a long time, most of our knowledge of gp340-microbial interactions is from studies with oral bacteria. For example, viridans streptococci, such as Streptococcus mutans and Streptococcus gordonii, interact with saliva gp340 via their surface proteins AgI/II and the sialic acid-binding Hsa or GspB adhesin. In these interactions, gp340 shows a peculiar fluid phase versus surface-adsorbed behavior, as evidenced by AgI/II polypeptides primarily mediating aggregation of bacteria by fluid phase gp340, whereas the Hsa adhesin primarily mediates adhesion of S. gordonii to surface-bound gp340 (18).gp340 binds also to many non-oral human Gram-negative and Gram-positive pathogens, such as Helicobacter pylori and S. pyogenes (10), but these interactions are less characterized. There are few studies suggesting that both carbohydrates and the protein core of the gp340 can be involved in these interactions. For example, a VEVLXXXXW peptide derived from the SRCR domain of gp340 is shown to bind different types of bacteria (19), whereas sialic acid residues may mediate binding to, for example, influenza virus (20). However, the molecular basis of the ability of gp340 or peptides derived thereof to bind a large ligand repertoire is not understood.The aim of the present study was to use the common human pathogen S. pyogenes as a model bacterium to identify novel bacterial proteins binding to gp340 and in this way shed light on the ligand recognition capability of gp340. We report a novel S. pyogenes-host interaction mediated by bacterial surface protein Spy0843 and gp340. gp340 binds to conserved leucine-rich repeat (Lrr) motifs in the Spy0843 and recognizes the same motif also in other bacterial proteins, from both Gram-negative and Gram-positive bacteria. Moreover, the inhibition of Lrr binding with SRCR-derived peptides differed from that previously reported, which suggests a novel mode for ligand recognition of human scavenger receptor gp340. |
