The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis.

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Glutamate-pantothenate pathway promotes antibiotic resistance of Edwardsiella tarda.

Although cellular metabolic states have been shown to modulate bacterial susceptibility to antibiotics, the interaction between glutamate (Glu) and chloramphenicol (CAP) resistance remains unclear because of the specificity of antibiotics and bacteria. We found that the level of Glu was upregulated in the CAP-resistant strain of Edwardsiella tarda according to a comparative metabolomics approach based on LC-MS/MS. Furthermore, we verified that exogenous metabolites related to Glu, the tricarboxylic acid (TCA) cycle, and glutathione (GSH) metabolism could promote CAP resistance in survival assays. If GSH metabolism or the TCA cycle is inhibited by L-buthionine sulfoximine or propanedioic acid, the promotion of CAP resistance by Glu in the corresponding pathway disappears. According to metabolomic analysis, exogenous Glu could change pantothenate metabolism, affecting GSH biosynthesis and the TCA cycle. These results showed that the glutamate-pantothenate pathway could promote CAP resistance by being involved in the synthesis of GSH, entering the TCA cycle by direct deamination, or indirectly affecting the metabolism of the two pathways by pantothenate. These results extend our knowledge of the effect of Glu on antibiotic resistance and suggest that the potential effect, which may aggravate antibiotic resistance, should be considered before Glu and GSH administration in the clinic.

Metabolites in the TCA Cycle Promote Resistance to Chloramphenicol of Edwardsiella tarda.

Antibiotic-resistant bacteria are a serious threat to human and animal health. Metabolite-enabled eradication of drug-resistant pathogens is an attractive strategy, and metabolite adjuvants, such as fumarate, are used for restoring the bactericidal ability of antibiotics. However, we show that metabolites in the TCA cycle increase the viability of Edwardsiella tarda against chloramphenicol (CAP), based on the survival assay of differential metabolites identified by LC-MS/MS. Furthermore, NADPH promotes CAP resistance in the CAP-resistant strain, while oxidants restore the bactericidal ability. Finally, we show that the intracellular redox state determines the sensitivity to CAP, and the total antioxidative capacity is decreased significantly in the antibiotic-resistant strain. Considering that the metabolites promote CAP resistance, metabolite adjuvants should be applied very cautiously. Overall, our research expands on the knowledge that the redox state is related to the bactericidal ability of CAP.