Significance of Glutamate Racemase for the Viability and Cell Wall Integrity of Streptococcus iniae.

Streptococcus iniae is a pathogenic and zoonotic bacterium responsible for human diseases and mortality of many fish species. Recently, this bacterium has demonstrated an increasing trend for antibiotics resistance, which has warranted a search for new approaches to tackle its infection. Glutamate racemase (MurI) is a ubiquitous enzyme of the peptidoglycan synthesis pathway that plays an important role in the cell wall integrity maintenance; however, the significance of this enzyme differs in different species. In this study, we knocked out the MurI gene in S. iniae in order to elucidate the role of glutamate racemase in maintaining cell wall integrity in this bacterial species. We also cloned, expressed, and purified MurI and determined its biochemical characteristics. Biochemical analysis revealed that the MurI gene in S. iniae encodes a functional enzyme with a molecular weight of 30 kDa, temperature optimum at 35°C, and pH optimum at 8.5. Metal ions, such as Cu2+, Mn2+, Co2+ and Zn2+, inhibited the enzyme activity. MurI was found to be essential for the viability and cell wall integrity of S. iniae. The optimal growth of the MurI-deficient S. iniae mutant can be achieved only by adding a high concentration of D-glutamate to the medium. Membrane permeability assay of the mutant showed an increasing extent of the cell wall damage with time upon D-glutamate starvation. Moreover, the mutant lost its virulence when incubated in fish blood. Our results demonstrated that the MurI knockout leads to the generation of S. iniae auxotroph with damaged cell walls.

Identification and initial functional characterization of lysosomal integral membrane protein type 2 (LIMP-2) in turbot (Scophthalmus maximus L.).

The immune system protects organism from external pathogens, this progress starts with the pathogen recognition by pattern recognition receptors (PRRs). As a group of PRRs, the class B scavenger receptors showed important roles in phagocytosis. Among three class B scavenger receptors, lysosomal integral membrane protein type 2 (LIMP-2) was reported to present in the limiting membranes of lysosomes and late endosomes, but its immune roles in teleost species are still limited in handful species. Here, we characterized LIMP-2 gene in turbot, and its expression patterns in mucosal barriers following different bacterial infection, as well as ligand binding activities to different microbial ligands and agglutination assay with different bacteria. In our results, one SmLIMP2 gene was identified with a 1,593 bp open reading frame (ORF). The multiple species comparison and phylogenetic analysis showed the closest relationship to Paralichthys olivaceus, the genomic structure analysis and syntenic analysis revealed the conservation of LIMP-2 during evolution. In tissue distribution analysis, SmLIMP-2 was expressed in all the examined turbot tissues, with the highest expression level in brain, and the lowest expression level in liver. In addition, SmLIMP-2 was significantly up-regulated in all the mucosal tissues (skin, gill and intestine) following Gram-negative bacteria Vibrio anguillarum infection, and was only up-regulated in gill following Gram-positive bacteria Streptococcus iniae challenge. Finally, the rSmLIMP-2 showed strong binding ability to all the examined microbial ligands, and strong agglutination with Escherichia coli, Staphylococcus aureus and V. anguillarum. Taken together, our results suggested SmLIMP-2 played important roles in fish immune response to bacterial infection. However, further functional studies should be carried out to better characterize its detailed roles in teleost immunity.

cpsJ gene of Streptococcus iniae is involved in capsular polysaccharide synthesis and virulence.

The capsular polysaccharides are an important virulence factor of Streptococcus iniae, protecting the bacterium from destruction and clearance by the immune system. The cpsJ gene encodes a putative UDP-glucose epimerase involved in the capsule synthesis system. To determine the role of the CpsJ protein in the production of the capsule, a ΔcpsJ mutant was generated and analyzed by comparing its growth performances and virulence with those of the wild type (WT) strain. The ΔcpsJ mutant had longer chains, smaller colonies, and a slower growth rate and decreased optical density than the WT, suggesting that the ΔcpsJ mutant produces less capsular polysaccharide. The ΔcpsJ mutant was more able to adhere to and invaded epithelioma papulosum cyprinid cells (EPCs) when its virulence in vitro was compared with that of the WT, but survived less well in the whole blood of channel catfish. When a channel catfish infection model was used to determine the virulence of the ΔcpsJ mutant in vivo, the mutant caused an increase in survival with the mutant (53.33 %) versus the WT (26.67 %). In summary, mutation of the cpsJ gene influenced both the capsule synthesis and virulence of S. iniae.

[Hyaluronic acid production by Streptococcus iniae and its application in rabbit skin's regeneration].

Hyaluronic acid (HA) is an important biomaterial as the extracellular matrix in human body. We produced HA by fermentation of Streptococcus iniae (Strep.). Production of HA by Strep. was evaluated and further improved by strain mutation by ultraviolet. One strain with higher HA yield and lower content of protein was obtained. Its HA yield increased from (82.3±3.3) mg/L to (120±10.6) mg/L, and protein decreased from (0.178±0.011) mg/L to (0.032±0.017) mg/L. The molecular weight (MW) of HA yield from Strep. is about 3.0×105 Da. Using the method of freezing and thawing, HA aqueous solution was transferred into hydrogel. This HA hydrogel, casted on sterilized non-woven fabric, was applied to repair rabbit skin with full-thickness defect. The preliminary results of the animal tests displayed that HA hydrogel obviously reduced the inflammation around the wound and promoted the skin regeneration comparing with the control tests.