Largemouth bass galectin, MsGal-9: Mediating various functions as a pattern recognition receptor and a potential damage-associated molecular pattern.

Galectins are lectins that bind to ß-galactose and are widely expressed in immune system tissues, playing pivotal roles in innate immunity through their conserved carbohydrate-recognition domains (CRDs). In this present investigation, a tandem-repeat galectin was discovered in the largemouth bass, Micropterus salmoides (designated as MsGal-9). The open reading frame of MsGal-9 encodes two CRDs, each containing two consensus motifs that are essential for ligand binding. MsGal-9 is expressed in various tissues of the largemouth bass, with particularly high expression levels in the liver and spleen. The full-length form of MsGal-9, as well as the N-terminal (MsGal-9-N) and C-terminal (MsGal-9-C) CRDs, were individually recombined. Their ability for nonself recognition was studied. The three recombinant proteins were able to bind to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS), with MsGal-9 displaying the highest binding activity. Furthermore, rMsGal-9-N exhibited higher binding activity towards GLU in comparison to rMsGal-9-C. Further investigations revealed that the full-length rMsGal-9 could significantly bind to Gram-positive bacteria, Gram-negative bacteria, and fungi, while rMsGal-9-C specifically bound to Escherichia coli. However, rMsGal-9-N did not exhibit significant binding activity towards any microbes. These findings indicate that MsGal-9 requires both CRDs to cooperate in order to fulfill its nonself recognition function. All three recombinant proteins demonstrated agglutination activity towards various microbes, with MsGal-9 and MsGal-9-N displaying a similar broad binding spectrum, while MsGal-9-C agglutinated three types of bacteria. Moreover, both MsGal-9 and MsGal-9-N were capable of coagulating largemouth bass red blood cells, whereas MsGal-9-C lacked this ability. However, MsGal-9-C played a significant role in enhancing the encapsulation of leukocytes in comparison to MsGal-9-N. All three proteins acted as potential damage-associated molecular patterns (DAMPs), inducing apoptosis in leukocytes.

Peptidoglycan recognition protein MsPGRP in largemouth bass (Micropterus salmoides) mediates immune functions with broad nonself recognition ability.

Peptidoglycan (PGN) recognition proteins (PGRPs) are important immune factors in innate immunity that function in recognising pathogens and activating the immune system. These ubiquitous proteins are conserved in invertebrates and vertebrates. In this study, a PGRP gene (MsPGRP) from largemouth bass (Micropterus salmoides) was identified and characterised, and its transcription distribution was explored. Recombinant protein (rMsPGRP) exhibited dose-dependent binding to PGN and glucan (GLU), but weak binding to lipopolysaccharide (LPS). MsPGRP exhibited agglutinating activity against several Gram-negative bacteria, Gram-positive bacteria and fungi, and it promoted phagocytosis activity of leukocytes against Micrococcus luteus and Aeromonas hydrophila. The protein also possessed amidase activity in the presence of Zn2+, degraded PGN, and disrupted the M. luteus cell wall. The results suggest that MsPGRP plays an important role in pathogen recognition, and acts as a opsonin during immune system responses and elimination of invading pathogens.

The role of TNF-α in the phagocytosis of largemouth bass (Micropterus salmoides) leukocytes.

Phagocytosis is an important innate immune process in which immune cells recognize, ingest and eliminate pathogens. Largemouth bass (Micropterus salmoides) has become an important economic farmed fish in many regions, while few studies has focused on phagocytosis of its leucocytes. In present study, largemouth bass peripheral blood leucocytes were separated using Percoll gradient to establish the phagocytic function. Flow cytometric analysis showed that largemouth bass leukocytes exhibited the phagocytic capacity to fluoresbrite microspheres and Aeromonas hydrophila, where higher phagocytic capacity to A. hydrophila were observed in granulocytes/monocytes than that of lymphocytes. The leukocytes engulfing fluoresbrite microspheres and A. hydrophila were also observed by fluorescence microscopy. Besides, manygenes associated with phagocytosis and TNF-α in leukocytes were up-regulated following A. hydrophila stimulation. Subsequently, the largemouth bass TNF-α was recombinantly expressed to investigate its role in regulating phagocytosis. The results showed that TNF-α in largemouth bass could significantly enhance the phagocytic ability of granulocytes/monocytes to A. hydrophila, but not lymphocytes. Moreover, we also found that TNF-α could not only significantly increase the ROS activity of granulocytes/monocytes, but also had the function of inducing its apoptosis. These results demonstrated that granulocytes/monocytes play more important role in phagocytosis, meanwhile, TNF-α has the function of enhancing the phagocytic ability of granulocytes/monocytes in largemouth bass.