Duck Complement Factor H Binds to Outer Membrane Protein Omp24 of Riemerella anatipestifer.

The resistance to serum complement-mediated killing is a vital virulence property of microbial pathogens. Complement factor H (FH) is a key negative regulator of the complement alternative pathway (AP) that prevents formation and accelerates the decay of AP C3 convertase and acts as a cofactor in the inactivation of C3b. Pathogens can recruit host FH through their surface proteins to escape the clearance of the complement system. Riemerella anatipestifer could also evade the complement system attack to achieve host infection, but the mechanism is still unclear. In this study, the R. anatipestifer proteins that could interact with FH in host serum were screened and analyzed, and the functions were determined. Affinity chromatography with a Ni-nitrilotriacetic acid Sefinose column and mass spectrometry identified three outer membrane proteins (Omp) of R. anatipestifer, Omp54, Omp53, and Omp24, as potential FH-binding proteins. We then successfully conducted the prokaryotic expression and polyclonal antibody preparation of three candidate proteins. Indirect immunofluorescence assay showed that three candidate proteins were all present in R. anatipestifer. The affinity blotting assay, anti-serum-inhibiting assay, and serum bactericidal assay presented evidence that Omp24 could bind FH. Moreover, FH bound to Omp24 was associated with resistance to the alternative pathway and functional for R. anatipestifer survival in the normal duck serum. These results suggested that R. anatipestifer Omp24 was a FH-binding protein and the interaction with FH blocked the alternative pathway. Recruitment of complement regulatory proteins may facilitate better R. anatipestifer resistance to this vital line of host defense.

Artículo regular­El factor H del complemento de pato se une a la proteína de la membrana externa Omp24 de Riemerella anatipestifer La resistencia a la destrucción mediada por el complemento sérico es una propiedad vital para la virulencia de los patógenos microbianos. El factor de complemento H (FH) es un regulador negativo clave de la vía alterna del complemento (AP) que previene la formación y acelera la descomposición de la C3 convertasa de la vía alterna y actúa como cofactor en la inactivación de C3b. Los patógenos pueden reclutar factor H del huésped a través de sus proteínas de superficie para escapar de la destrucción por el sistema del complemento. Riemerella anatipestifer también pudo evadir el ataque del sistema del complemento para lograr la infección del huésped, pero el mecanismo aún no está claro. En este estudio, se seleccionaron y analizaron las proteínas de R. anatipestifer que podrían interactuar con el factor H en el suero del huésped y se determinaron las funciones. La cromatografía de afinidad con una columna de sefinosa de Ni-NTA y la espectrometría de masas identificaron tres proteínas de la membrana externa de R. anatipestifer, Omp54, Omp53 y Omp24, como posibles proteínas que se unen al factor H. Posteriormente, se llevó a cabo con éxito la expresión procariota y la preparación de anticuerpos policlonales de las tres proteínas candidatas. El ensayo de inmunofluorescencia indirecta mostró que las tres proteínas candidatas estaban presentes en R. anatipestifer. El ensayo de transferencia para afinidad, el ensayo anti-inhibidor del suero y el ensayo bactericida sérico presentaron evidencia de que la proteína Omp24 podría unirse al factor H. Además, el factor H unido a la proteína Omp24 se asoció con resistencia a la vía alterna y funcional para la supervivencia de R. anatipestifer en el suero de pato normal. Estos resultados sugirieron que la proteína Omp24 de R. anatipestifer era una proteína de unión al factor H y que la interacción con este factor bloqueaba la vía alterna del complemento. El reclutamiento de proteínas reguladoras del complemento puede facilitar una mejor resistencia de R. anatipestifer a esta línea vital de defensa del huésped.

[Cloning and expression of duck C4BPα and verification of its interaction with Riemerella anatipestifer].

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.