Neu1 deficiency increases the susceptibility of zebrafish to Edwardsiella piscicida infection via lysosomal dysfunction.

Neu1 is a lysosomal glycosidase that catalyzes the removal of sialic acids from glycoconjugates. Although Neu1 sialidase is highly conserved among vertebrates, the role of fish Neu1 is not fully understood because of its unique aquatic living situation. Compared to land animals, fish have a higher chance of bacterial infection, and to understand the role of fish Neu1, the susceptibility of Neu1 knockout zebrafish (Neu1-KO) was evaluated using Edwardsiella piscicida, a fish pathogen. Neu1-KO larvae showed high susceptibility to E. piscicida, despite the activation of macrophages, and presented increased lysosomal signals induced by the accumulation of Sia α2-3 linked oligosaccharides. The accumulation coincided with the signal of the macrophage marker, suggesting that the dysfunction of lysosomes in macrophages would result in a high susceptibility of Neu1-KO to E. piscicida. Chloroquine, an inhibitor of lysosomal degradation, induced high mortality of wild type zebrafish with E. piscicida infection accompanied by increased lysosomal accumulation, similar to Neu1-KO zebrafish. This study revealed that Neu1 sialidase plays a crucial role in the lysosomal degradation of macrophages with a bacterial infection.

Enhancement of Edwardsiella piscicida infection, biofilm formation, and motility caused by N-acetylneuraminate lyase.

Sialic acid and its catabolism are involved in bacterial pathogenicity. N-acetylneuraminate lyase (NAL), which catalyzes the reversible aldol cleavage of sialic acid to form N-acetyl-D-mannosamine in the first step of sialic acid degradation, has been recently investigated to elucidate whether NAL enhances bacterial virulence; however, the role of NAL in bacterial pathogenicity remains unclear. In the present study, we demonstrated that the existence of two enzymes in Edwardsiella piscicida, referred to as dihydrodipicolinate synthase (DHDPS) and NAL, induced the cleavage/condensation activity toward sialic acids such as N-acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid. NAL enhanced cellular infection in vitro and suppressed the survival rate in zebrafish larvae in bath-infection in vivo, whereas DHDPS did not. Furthermore, NAL strongly activated the expression of E. piscicida phenotypes such as biofilm formation and motility, whereas DHDPS did not. Besides, the gene expression level of nanK, nanE, and glmU were up-regulated in the NAL-overexpressing strain, along with an increase in the total amount of N-acetylglucosamine.

Host lactosylceramide enhances Edwardsiella tarda infection.

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-ß-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.