Metabolic Alterations in Shrimp Stomach During Acute Hepatopancreatic Necrosis Disease and Effects of Taurocholate on Vibrio parahaemolyticus

Abstract

Acute hepatopancreatic necrosis disease (AHPND), a recently emerged bacterial shrimp disease, has increased shrimp mortality and caused huge economic losses in many Asian countries. However, molecular factors underlying pathogenesis of this disease remain largely unknown. Our objective was to characterize metabolic alterations in shrimp stomach during AHPND and determine effects of taurocholate on AHPND-causing Vibrio parahaemolyticus. Based on metabolomics, pathways for lipid metabolism and for primary bile acid (BA) synthesis were majorly affected following AHPND infection. Bile acid metabolites, namely taurocholate, were downregulated in the metabolomics database. This prompted us to study effects of taurocholate on biofilm formation, PirAB ^vp toxin release and biofilm detachment capabilities in AHPND-causing V. parahaemolyticus. Treatment of this bacterium with high concentration of taurocholate, a primary bile acid, induced biofilm formation, PirAB ^vp toxin release and facilitated the dispersion of bacterial cells. Taken together, our findings suggest that AHPND infection can affect the lipid metabolites in shrimp stomach, and further suggest that the primary bile acid taurocholate is important for the virulence of AHPND-causing V. parahaemolyticus.

Keywords: AHPND; Vibrio parahaemolyticus; lipid metabolism; metabolomics; shrimp; taurocholate.

Figure 1

Overview of metabolome profiling assembly pipeline and principal component analysis (PCA) of differentially expressed metabolites (DEMs). (A) Acute hepatopancreatic necrosis disease (AHPND)-causing strain (5HP)-infected shrimp stomach samples were used for metabolomic profiling using UHPLC-QTOF-MS. In total, 2,725 and 2,614 metabolites were obtained in positive (POS) and negative (NEG) ion modes, respectively. (B) Principle component analysis of DEMs from samples collected from quality control (QC), tryptic soy broth (TSB), non-AHPND-causing strain (S02), and 5HP groups. The 18 pooled samples and three QCs analyzed in this study were classified into two distinct clusters in both POS and NEG ion modes. The PCA analysis also identified TSB#4 as an outlier; this sample was excluded from further analyses.

Figure 2

The strategy of data filtering for shrimp metabolites. Comparison of DEMs in both POS ion and NEG ion modes of 5HP vs. TSB and 5HP vs. S02 at 12 hours post infection (hpi). (A) In POS ion mode, from the 2,725 metabolites, there was at least one 5HP, TSB, or S02, 503 DEMs with a 2-fold change (p < 0.05). (B) Similarly, from the 2,164 metabolites in NEG ion mode, 634 DEMs with a 2-fold change and p < 0.05 were obtained. The DEMs were further annotated using proprietary MS1, MS2 database, and Kyoto encyclopedia of genes and genomes (KEGG).

Figure 3

Functional classification of DEMs according to their corresponding pathways. (A) Proportion of upregulated and downregulated metabolites associated with lipid metabolism of 5HP-infected shrimps in POS and NEG ion modes. (B) KEGG pathway breakdown of the lipid metabolism DEMs. Metabolites without MS/MS annotations were excluded.

Figure 4

Acute hepatopancreatic necrosis disease infection affected the expression of genes related to the biosynthesis of unsaturated fatty acids. Stomach samples of 5HP-challenged and S02-challenged shrimps were collected at (A) 12 and (B) 24 hpi were analyzed for expression of genes involved in the biosynthesis of unsaturated fatty acids (LvACT: Acyl-coenzyme A thioesterase, LvPPT: Palmitoyl-protein thioesterase, LvΔ-9 Desaturase). Graphs represent mean ± SD. Differences between treatment groups are indicated by asterisks (Student’s t-test, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001).

Figure 5

Expression of bile acid synthesis genes was modulated during AHPND infection. Stomach samples collected at (A) 12 and (B) 24 hpi from shrimp challenged with either S02 or 5HP were used to analyze gene expression of bile acid synthesis related genes (LvACOX, LvAMACR, LvBAAT, LvPMFE, and LvSCP). Data are mean ± SD. Differences between treatment groups are indicated by asterisks (Student’s t-test, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001).

Figure 6

Taurocholate induced biofilm formation in AHPND-causing Vibrio parahaemolyticus. Biofilm assays were conducted at various concentrations of taurocholate for (A) M1-1, (B) 5HP, (C) S02, (D) Escherichia coli, and (E) Staphylococcus aureus in 96-well plates. The biofilms were stained with crystal violet stain and quantified at OD₅₅₀nm. The data represent fold change compared to no-bile-acid control. Each bar represents mean ± SD. The statistically significant differences between treatment and control groups are indicated as asterisks (Student’s t-test, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001).

Figure 7

Taurocholate increased the number of both biofilm and planktonic AHPND-causing V. parahaemolyticus 5HP in culture supernatant. Effect of taurocholate on the number of planktonic bacterial cells was assessed by treating the AHPND-causing V. parahaemolyticus 5HP with crude bile acids and taurocholate. (A) Biofilm formation in the presence of taurocholate.The data represent fold change compared to no-bile-acid control. Each bar represents mean ± SD. The statistically significant differences between the treatment groups and the no-bile-acid control group are indicated as asterisks (Student’s t-test, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001). (B) Proportion of planktonic and biofilm cells.

Figure 8

Taurocholate stimulated PirAB^vp toxin release in AHPND-causing V. parahaemolyticus. Effects of taurocholate on release of PirA^vp and PirB^vp toxins were assessed by Western blots. Toxin concentrations were detected in the pellet (P) and supernatant (S) of AHPND-causing V. parahaemolyticus 5HP strain cultured with taurocholate for (A) 3 or (B) 16 h. Immunoblots were probed with PirA^vp and PirB^vp antibodies, respectively. The intensities of each pair of immunoblots are shown graphically as relative expression in supernatant (Sup) and pellet compared to their corresponding no-bile-acid control.