Recombinant sialidase NanA (rNanA) cleaves alpha 2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection

摘要

Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, alpha 2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in alpha 2-3 sialo-linked glycoproteins, but not in glycolipids and alpha 2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that alpha 2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of alpha 2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of alpha 2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAMS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing chains following desialylation. Together, these results suggest that E. tarda may employ endogenous NanA to desialylate alpha 2-3 glycoproteins on host cells, thus revealing one of the potential binding molecules during infection. (C) 2015 Elsevier Ltd. All rights reserved.