Metabolomics:Open Access

Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification

Abstract

Gao D, Liu N, Li Y, Zhang Y, Liu G, Xiao G and Lu C

Eha is a virulence regulator associated with the replication of Edwardsiella tarda within RAW264.7 macrophages. Eha is required for the bacteria to resist the acid and oxidative stress in macrophages. We herein demonstrate that Eha regulates the resistance of this bacterium against acidification in macrophages and explain the underlying molecular mechanism. Firstly, to find an acid or oxidative condition to induce the strongest Eha activities, we constructed pMP220- Pehalac Z plasmid and inspected the lac Z expression regulated by Eha by using a β-galactosidase assay. At exposure of pH6.3 medium 2 h, whole transcriptomic profiling of the wild type and mutant were performed by RNA-sequencing. We identified 147 differentially -expressed genes (DEGs) (|log2 ratio| ≥ 1), 113 and 34 of which were significantly up- and down-regulated, respectively, in the mutant compared with the wild type. These findings were validated by qRT-PCR. A CO functional analysis revealed that these genes were divided into 25 categories, including the bacterial processing, localization, metabolism, combination, catalysis, transportation and cellular composition. Based on the KEGG database, these genes were distributed in 55 pathways, such as the two-component system, ABC transporters, and microbial metabolism. At last, the intracellular survival rates and intraphagosomal pH of wild type ET 13 and its eha mutant in bafilomycin-treated and untreated macrophages were measured. The experiment showed that Eha was involved in protecting the bacteria from the effects of acidification within macrophages. The survival rate of the wild was also higher than that of the mutant under acid stress both in vivo and in vitro (P<0.05). Overall, Eha was found to regulate 147 genes that affect bacterial metabolism and virulence, allowing the bacteria to adapt to an acidic environment. These results could be helpful for further investigations of the mechanisms by which Edwardsiella tarda survives in macrophages.