Targeted metabolomics to investigate antimicrobial activity of itaconic acid in marine molluscs.

Abstract

Itaconic acid (ITA) has recently been identified as an antimicrobial metabolite in mammalian immune cells. The presence of ITA was alsoreported in different tissues of marine molluscs, indicating its role as an endogenous metabolite of molluscs. In addition, the accumulation of ITA has been observed in different tissues of mussels following pathogen challenges. However, the concentration of ITA in mussel tissues and the possible role of this metabolite in the molluscan innate immune system remain unknown. This study aims to quantitatively measure ITA levels in different tissues of marine mussels following an experimental challenge with Vibrio sp. DO1 isolate, and to identify the antimicrobial role of ITA in the innate immune system through the measurement of metabolic and immune alterations in tissues following the challenge. In this study, adult Perna canaliculus mussels were experimentally challenged with a pathogenic Vibrio sp. DO1 isolate. The metabolite profiles of five different tissues, including mantle, gill, muscle, hepatopancreas and haemolymph were obtained, and levels of ITA in each tissue were characterized using a gas chromatography-mass spectrometry (GC-MS) metabolomics approach. Flow cytometry was also employed to measure cell health parameters, including oxidative stress via reactive oxygen species (ROS) production, apoptosis via changes in mitochondrial membrane potential (MMP) and haemocyte viability. The ITA levels in mantle, gill, muscle and hepatopancreas tissues at 18-h post infection (hpi) with Vibrio sp. were 40.31, 41.71, 11.61 and 41.66ngmg-1, respectively. In haemolymph, the level of ITA was significantly increased from 95.25ngml-1 at 6 hpi to 174.36ngml-1 at 18hpi and 572.12ngml-1 at 60hpi. In line with the accumulation of ITA, we observed increased levels of metabolites within the tricarboxylic acid (TCA) cycle, anti-inflammatory metabolites and alterations of other metabolites associated with immune responses of the host. The flow cytometry analyses revealed increases in ROS production, apoptotic cells and decreases in cell viability. We reported on the production of ITA in different tissues of P. canaliculus mussels challenged with a marine pathogen which confirmed ITA as an antimicrobial metabolite. The findings revealed insights into the biosynthesis of ITA and suggests its role in antimicrobial and anti-inflammatory activities in the innate immune system. This study also provided insights into the innate immune system of bivalves and highlighted the potential use of ITA as a biomarker for shellfish health assessment in aquaculture.