Probiotic, Bacillus subtilis E20 alters the immunity of white shrimp, Litopenaeus vannamei via glutamine metabolism and hexosamine biosynthetic pathway

Highlights

• Creatinine and glutamine (Gln) increased in the hepatopancreas of shrimp fed a diet containing Bacillus subtilis E20.

• B. subtilis E20 promotes the gene expressions that are associated with hexosamine biosynthesis pathway (HBP), and protein O-glycosylation in shrimp.

• B. subtilis E20 improves shrimp health status via the Gln to increase HBP and protein O-glycosylation.

Abstract

The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 10⁹ cfu kg diet⁻¹. After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using ¹H nuclear magnetic resonance (¹H NMR) based metabolomic analysis. Results from the ¹H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analysis. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp.

Introduction

The interactions between microbiota and hosts (including shrimp) begin at birth, and the intestinal microbiota are known to affect the host's health [1,2]. The diversity of the microbiota is dependent on feeding as well as on various environmental factors [[2], [3], [4]]. For example, white shrimp, Litopenaeus vannamei that were fed synbiotic (Lactobacillus plantarum plus galacto-oligosaccharide) showed greater colonization of Lac. plantarum and lower potential pathogens in the intestines (Photobacterium damselae and Vibrio harveyi) [2]. Similarly, the intestinal bacterial population of white shrimp changed significantly and the beneficial bacteria increased after receiving dietary probiotics [5]. Maintenance of a healthy microflora in animal intestines can extend lifespan and prevent disease [6].

Probiotics have been used to improve health condition of aquacultural animals by regulating the immune system, providing resistance against infections, and improving nutrient utilization and host metabolism [[7], [8], [9], [10], [11], [12], [13]]. Some Bacillus are considered to be promising probiotics which are able to inhibit colonization of potential pathogens in intestine of shrimp, thereby boosting shrimp immunity and disease resistance [[14], [15], [16], [17]]. Probiotics can increase nutrient utilization in shrimp [10]. The increased feed digestibility might explain why shrimp growth improved [8,9,18]. Although the efficiencies of probiotic in the improvement of shrimp growth and health status are widely evaluated, the mechanisms responsible for these effects have not been fully elucidated.

Amino acids (AA) have been classified as essential AA and non-essential AA for animals. In general, animals can synthesize sufficient amounts of non-essential AA and do not need them in diets for optimal nutrition or health. However, growing evidence from cell culture and animal studies shows that some of the non-essential AA, such as glutamine (Gln) play an important role in physiological regulation [19] and some evidence has shown that Gln supplementation is very important for surgical patients to modulate postoperative immune and inflammatory response following portal hypertension surgery [20]. The importance of Gln in cell survival and proliferation was first described by Ehrensvard et al. [21]. Glutamine is related to different metabolism pathways, such as the nitrogen donor for the synthesis of nucleic acids, as precursor to AA synthesis, as energy substrate, contribution to the acid-base balance, and aiding in the synthesis of intracellular protein [22]. Glutamine is also known to be an immune-nutrient in regulating the immune responses; serving as fuel for many cells including lymphocytes, macrophages, fibroblasts in culture and malignant cells [23], promoting heat shock protein release [24], and involving the regulation of inflammatory response [25,26]. Since nutrients have a variety of bio-functions, the increased digestibility of nutrients in shrimp via the dietary probiotic might contribute to the modulation of physiology including immune-modulation.

RNA-seq is a technique with high sensitivity for identifying differentially expressed genes and has been applied in whole-genome analysis of nutrition studies of aquatic animals [27,28]. By using the RNA-seq, a far more precise measurement of transcripts under different conditions can be obtained and a general view of gene expression can be shown, especially in the absence of a fully sequenced and assembled animal genome. In addition to high-throughput sequencing, metabolomics is another emerging field and is generally acknowledged as the comprehensive measurement of low-molecular-weight molecules and all metabolites in a biological specimen. Metabolomics is able to profile more metabolites than the standard clinical laboratory techniques, and can comprehensively cover biological processes and metabolic pathways. The ¹H nuclear magnetic resonance (NMR) was applied for the metabolomics analysis of synbiotic-fed shrimp, and the results clearly indicated that some metabolites, such as inosine monophosphate, valine, and betaine related to the increased defense capacity of haemocytes such as triggering of melanization and phagocytosis processes, and resulted in enhanced immunity and disease resistance of shrimp [29].

In this study, two powerful tools, RNA-seq and ¹H NMR-based metabolomics analysis were applied to a comprehensive survey of transcripts and metabolites associated with immune system of probiotic, B. subtilis E20-fed shrimp. This systematic analysis of transcriptomes and metabolomics may facilitate further understanding of the molecular mechanism of immune regulation of probiotic-fed shrimp.