Bile acid and bile acid transporters are involved in the pathogenesis of acute hepatopancreatic necrosis disease in white shrimp Litopenaeus vannamei.

Acute hepatopancreas necrosis disease is a recently emerged shrimp disease that is caused by virulent strains of Vibrio parahaemolyticus. Although AHPND poses a serious threat to the shrimp industry, particularly in Asia, its underlying pathogenic mechanisms are not well characterized. Since a previous transcriptomic study showed upregulation of the apical sodium bile acid transporter (LvASBT), our objective here was to explore the role of bile acids and bile acid transporters in AHPND infection. We confirmed that mRNA expression of LvASBT was upregulated in the stomach of AHPND-infected shrimps. Bile acid concentrations were also higher in the stomach of AHPND-infected shrimp and correlated with high expression of pVA plasmid and Pir toxins. In vitro assays showed that bile acids enhanced biofilm formation and increased the release of PirABvp toxins in AHPND-causing V. parahaemolyticus, while in vivo inhibition of LvASBT by GSK2330672 reduced the copy numbers of pVA plasmid, Pir toxin and reduced the amounts of bile acids in AHPND-infected shrimp stomach. Transcriptomics data for AHPND-causing V. parahaemolyticus treated with bile acids showed upregulation of various genes involved in membrane transport, RND efflux pumps and a bacterial secretion system. Taken together, our results show that AHPND-causing V. parahaemolyticus virulence is positively regulated by bile acids and that LvASBT and bile acids in shrimp stomach have important roles in AHPND pathogenesis.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp.

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND-associated plasmid pVA1 encoding a virulent toxin (Pirvp ) that damages the shrimp's hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimp's stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND-induced changes in the stomach of AHPND-causing V. parahaemolyticus (5HP)-infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene-to-gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP-infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.

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

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.

Penaeus vannamei serine proteinase inhibitor 7 (LvSerpin7) acts as an immune brake by regulating the proPO system in AHPND-affected shrimp.

Acute hepatopancreatic necrosis disease (AHPND) is a recently emerged disease in aqua cultured shrimp that is caused by virulent strains of Vibrio parahaemolyticus (VP). Our previous study used transcriptomics to identify key pathogenic factors in the stomach of AHPND-infected shrimp (Litopenaeus vannamei), and here we used a different subset of the same data to construct a gene-to-gene expression correlation network to identify immune-responsive genes. LvSerpin7 was found to have the highest number of correlations after infection, and it also showed a significant increase in mRNA expression. LvSerpin7 is expressed in all tissues but its expression levels are highest in hemocytes. After successfully silencing LvSerpin7 transcript prior to AHPND challenge, mortality was significantly increased relative to the controls and reached 100% within 36 h post infection. Compared to the controls, the phenoloxidase (PO) activity also increased in both hemolymph and stomach. Recombinant LvSerpin7 inhibited shrimp PO activity in vitro, and we also found that rLvSerpin7 inhibited the growth of AHPND-causing bacteria. These results suggest that LvSerpin7 might reduce the toxic effects that result from unregulated activation of the PO defense system by AHPND-causing bacteria.

Draft Genome Sequence of Vibrio parahaemolyticus Strain M1-1, Which Causes Acute Hepatopancreatic Necrosis Disease in Shrimp in Vietnam.

We report here the genome sequence of Vibrio parahaemolyticus strain M1-1, which causes a mild form of shrimp acute hepatopancreatic necrosis disease (AHPND). Compared to other virulent strains, the M1-1 genome appeared to express several additional genes, while some genes were missing. These instabilities may be related to the reduced virulence of M1-1.