Therapeutic Effect And Mechanism Of Chitosangentamicin Conjugate On Shrimp Infected By Vibrio Parahaemolyticus

In recent years,with the development of shrimp aquaculture,the proportion of high-density intensive aquaculture has rapidly increased.It brings huge benefits,but easily leads to the deterioration of water quality and frequent outbreaks of bacteria and viruses.Among them,Vibrio parahaemolyticus is one of the main bacterial causing a large number of shrimp deaths.It can cause red body,rotten gills,white turbidity and acute hepatopancreas necrosis of shrimps,resulting in huge economic losses.Therefore,how to effectively prevent and control Vibrio parahaemolyticus has become the difficulty and focus of aquaculture.Chitosan（CS）is a linear polycationic polysaccharide obtained by deacetylating chitin,it has many biological properties and physiological functions.However,its antibacterial activity is weak and can only be dissolved in acidic solutions,limiting its application in the antimicrobial field.Therefore,modification of chitosan to improve its antibacterial activity and solubility has become the focus.Gentamicin（GT）has excellent antibacterial properties and water solubility due to its rich amino groups.However,the disadvantages of bacterial resistance,ototoxicity and nephrotoxicit,limit its applications.In this article,the polymer antibacterial agent chitosan-gentamicin conjugate（CS-GT）was prepared by grafting small molecule gentamicin onto the backbone of large molecule chitosan through oxidation,Schiff base and reduction.The goal is to improve the antibacterial activity and water solubility of chitosan,change the original antibacterial target of gentamicin and reduce its negative effects.On the other hand,the antibacterial effect of CS-GT on Vibrio parahaemolyticus,the microbial flora changes of cultured water and shrimp intestinal by16S r RNA gene sequencing,the analysis of RNA-seq high-throughput sequencing and the discussion of immune mechanism were also investigated.The purpose of this article is to provide a theoretical basis for prevention and control of Vibrio parahaemolyticus and healthy breeding of Litopenaeus vannamei.The main researches and results are as follows:1.The CS-GT was prepared by oxidation,Schiff base and reduction.It was characterized by FTIR,¹H-NMR and solid ¹³C-NMR.The synthesis mechanism of CS-GT was as follows:Firstly,chitosan was oxidized to chitosan dialdehyde by sodium periodate,then Schiff base reaction with gentamicin was occured,and finally C=N was reduced to C-N with sodium cyanoborohydride.The grafting rate of gentamicin was discussed through elemental analysis,the results showed that the main factors of the grafting rate are the sodium periodate dosages and oxidation time,it was 17.02%%in this article.The stability of conjugate before and after reduction was compared by thermal analysis,it was found that the reduction could improve the thermal stability of CS-GT,resulting in the weight loss rate was reduced by18.45%.2.The antibacterial effect of CS-GT on Vibrio parahaemolyticus was investigated,the results showed that the antibacterial activity of CS was significantly improved after grafting GT（p<0.05）.Moreover,the antibacterial activity of CS-GT was not no significant difference before and after reduction.The minimum inhibitory concentration（MIC）,minimum bactericidal concentration（MBC）and half-inhibitory concentration(IC₅₀)of CS-GT were 20.00,75.00 and 18.72μg/m L,respectively,they were much significantly lower than CS（p<0.01）.CS-GT could enhance the permeability of cell membrane,leading to the leakage of electrolytes（such as K⁺,Na⁺and H⁺）and macromolecules（DNA and RNA）in the cells,therefore,the conductivity,K⁺concentration and OD₂₆₀value were significantly increased（p<0.05）.Compared with CS,the positive charge density of CS-GT increased,resulting in the strengthening of electrostatic interaction with cell membrane,so its antibacterial activity,antibacterial and bactericidal ability were significantly improved（p<0.05）.3.The effects of CS-GT on aquaculture water and Litopenaeus vannamei were discussed in this article.Through the analyses of total hemocyte counts,hemocyte apoptosis,antioxidant and immune related enzyme activities,it was found that CS-GT significantly increased the number of total blood cells,antioxidant and immune related enzyme activities,inhibited the blood cell apoptosis,improved the survival rate of shrimps（p<0.05）.CS-GT increased the villi height of intestines,which is beneficial for absorption,digestion and utilization of food.The aquaculture water and intestinal flora of shrimps was analyzed by 16S r RNA gene high-throughput sequencing technology,it was found that CS-GT reduced the diversities and abundances of water and intestinal flora,its flora structure was similar to GT.CS-GT reduced the abundances of Vibrio and Photobacterium in water and intestines.Futhermore,the abundance of Vibrio was negatively correlated with CS-GT dose.4.The effect of CS-GT on immunity and intestinal flora of Litopenaeus vannamei infected with Vibrio parahaemolyticus was investigated.It was found that CS-GT significantly improved the survival rate,increased the total blood cell counts,decreased the blood cell apoptosis,enhanced the antioxidant and immune related enzyme levels of Litopenaeus vannamei infected with Vibrio parahaemolyticus（p<0.05）.In addition,they were in a CS-GT dependent mode.CS-GT could inhibit the pathogenic bacteria invasion and inflammatory reaction,protecting the structure and function of the intestine and hepatopancreas.The intestinal flora was analyzed through the 16S r RNA gene high-throughput sequencing technology,the results showed that CS-GT inhibited the invasion and colonization of Vibrio,significantly reduced the ratios of Firmicutes and Bacteroidetes（p<0.05）,improved the imbalance of intestinal flora,activated the relevant biological functions of immune system.5.The immune regulation of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was explored at the transcriptome level by RNA-seq high-throughput sequencing technology.The results showed that there were total 872 differentially expressed genes regulated by CS-GT,458 was up-regulated and 414 was down-regulated.The lysosome signaling pathway was significantly enriched.It was found that CS-GT inhibited the colonization and growth of Vibrio parahaemolyticus,regulated the structure of the intestinal flora,activated the immune-related biological functions,promoted the expressions of LGMN,LIPA and NPC2 up-regulated through the interaction between intestinal flora and cytokines.It is speculated that CS-GT will promote antigen presentation or hydrolytic activation of TOR-like receptors,participate in the acceleration process of triglyceride（TG）metabolism and the process of intracellular cholesterol transport,stimulating the lysosome signaling pathway to phagocytose pathogens more effectively,the body producing an immune response.