The control cells were added with PBS

The control cells were added with PBS. the presence of rCsCD46, tissue dissemination and survival of the pathogens were significantly inhibited. These results provide the first evidence to indicate that CD46 in teleosts negatively regulates complement activation via FI and protects host cells from complement-induced damage, and that CD46 is required for optimal bacterial infection probably by serving as a receptor for the bacteria. Introduction The complement system is a crucial constituent of the immune system and provides effective protection via mechanisms involving both innate and adaptive immune defenses1,2. The complement system consists of three pathways of activation: the classical pathway, the alternative pathway, and the lectin pathway3. The three pathways converge at the cleavage of C3 to C3a and C3b, which leads to a series of events involving cleavage of C5 and assembly of C5b, C6, C7, C8, and C9 to generate the membrane attack complex that induces osmotic lysis of the target cells4,5. Complement activation is regulated by many factors6. Factor I, a serine protease, is one of the complement regulatory proteins7. In humans, factor I regulates the complement activation cascades of both classical and alternative pathways by cleavage and degradation of C4b and C3b in the presence of certain cofactors, thereby preventing the assembly of the C3 and C5 convertases8C10. CD46 is usually a cell surface molecule that functions in monomeric form as a key regulator of the classical and PEPCK-C alternative complement activation cascades11. CD46 is usually a cofactor of factor I and regulates complement activation by facilitating the proteolysis of deposited C3b and C4b by factor I, thus preventing amplification of the complement cascade around the cells on which CD46 is expressed12,13. CD46 consists of four homologous complement control protein (CCP) repeats, a serine-threonine-proline-rich (STP) domain name, a transmembrane hydrophobic domain name, a cytoplasmic anchor, and a cytoplasmic tail14,15. In addition to its role in complement activation, CD46 BGJ398 (NVP-BGJ398) is also employed as a cellular receptor by several viruses and bacteria11. Human-specific pathogens targeting CD46 include measles virus, adenovirus groups B and D, herpes virus, (Fig.?1). CsCD46 also shared 25.7% overall sequence identity with human CD46, mainly in the CCP regions BGJ398 (NVP-BGJ398) (Fig.?1). Open in a separate window Physique 1 Alignment of the sequences of CsCD46 homologues. Dots denote gaps introduced for maximum matching. Numbers in brackets indicate overall sequence identities between CsCD46 and the compared sequences. The consensus residues are in red, the residues that are?75% identical among the aligned sequences are in grey. The signal peptide sequence is usually indicated by a black line, the CCP domain name is usually indicated by star signal, the transmembrane region is usually indicated by triangles, and the intracellular region is usually indicated by circles. The GenBank accession numbers of the aligned sequences are as follows: and are common fish pathogens. BGJ398 (NVP-BGJ398) Subsequent ELISA analysis showed that rCsCD46 exhibited apparent binding to in a dose-dependent manner (Fig.?5A). Relatively high binding indexes were observed with and and (data not shown). To examine whether rCsCD46 bound bacteria and CsFI via the same site in rCsCD46, was incubated with rCsCD46 in the presence or absence of rCsFI. Subsequent ELISA analysis showed that the presence of rCsFI had no significant effect on the binding index of rCsCD46 to bacteria (Fig.?5B). Open in a separate window Physique 5 Binding of rCsCD46 to bacteria. (A) were incubated with or without (control) different concentrations of rCsCD46, and bacteria-protein binding was determined by ELISA. (B) was incubated with or without (control) rCsCD46 plus rCsFI, rCsCD46 plus rTrx, rCsCD46, rCsFI, or rTrx (all proteins were at a final concentration of 40?g/ml), and protein-bacteria binding was determined as above..