Discovery and Structural Elucidation of Novel Microcystins Using MS and MSn with Python Code

Microcystins (MCs) are cyclic heptapeptide hepatotoxins with a vast structural diversity (\u3e300 congeners). However, many more congeners are theoretically possible, and a workflow was developed for putative identification of novel MCs using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) and MSn with Python code. Water samples collected from Lake Erie were sonicated and filtered, and solid-phase extraction was performed. A portion of the water sample was reacted with mercaptoethanol using the method developed by Miles et al. for structural elucidation. Extracted MCs were analyzed using UHPLC coupled to an Orbitrap Fusion instrument for HRMS. Collision-induced dissociation (CID) and higher-energy CID (HCD) were used for MSn analyses. A total of 33 MCs were found in lake water samples, including two unknown MCs. Two Python codes were developed to elucidate the structures of MCs. Code 1 was written to generate a list of masses of theoretically possible MC congeners. Code 2 was written to compare the experimentally obtained masses of MC fragment ions to the theoretical fragment masses. Using Codes 1 and 2, the two unknown MCs with m/z values of 526.7980 (z=+2) and 1025.5302 (z=+1) were putatively identified as MC-HarR and MC-E(OMe)R, respectively

New insights on organic nitrogen assimilation in Microcystis phycosphere and impacts on microcystin production

Microcystis is one of the most globally abundant bloom-forming cyanobacteria, comprising species that produce nitrogen-rich hepatotoxic compounds known as microcystins (MCs). Although dissolved organic nitrogen can exceed 50% of the nitrogen pool in aquatic ecosystems, organic nitrogen compounds are often overlooked in terms of their impact on community dynamics and Microcystis bloom development. Further, cyanobacteria are constantly interacting with other microorganisms in their surrounding environment, but nitrogen cycling in the phycosphere is poorly understood. In the present study, we monitored the growth and MC production of several Microcystis strains on various organic nitrogen sources including amino acids and proteins, investigated the impacts of organic nitrogen on Microcystis microbiome via amplicon and metagenomic sequencing, and traced the nitrogen assimilation within the phycosphere at the single-cell level by measuring isotopic tracer incorporation via secondary ion mass spectrometer imaging (NanoSIMS). We demonstrate that 1) organic nitrogen species shape the microbiome community structure in the Microcystisphycosphere, and 2) competition for and/or transport of nitrogen between heterotrophic bacteria and cyanobacteria potentially play important roles for cyanobacterial succession especially under inorganic nitrogen scarcity. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA2734

Cyanotoxin Degrading Lake Bacteria Significantly Alleviate Microcystin-LR Induced Hepatotoxicity in Both In Vitro and In Vivo Models

Our recent reports have shown that exposure to microcystin-LR (MC-LR) exacerbates the development of pre-existing liver and inflammatory bowel disease as well as alters gut microbiota that may significantly impact development of hepatotoxicity. We have isolated naturally occurring novel MC-LR degrading bacteria from Lake Erie, OH and hypothesized that they may alleviate MC-LR toxicity. qPCR analysis for markers of hepatotoxicity and inflammation in both in vivo and in vitro (using human Hep3B hepatocytes) settings showed significant downregulation in their expression in presence of MC degrading bacteria compared to the untreated groups. LC-MS analysis of the 24-hour urine samples in an in vivo setting with age matched Balb/c female mice that were pre-treated with the bacteria prior to 500 μg/kg MC-LR exposure for 24 hrs revealed significant reduction in urine MC-LR levels of mice pre-treated with MC-LR degrading bacteria as compared to the control group. Analysis of genes related to MC-LR induced apoptosis, DNA damage, ER stress, and fatty acid metabolism were also significantly downregulated in mice treated with MC degrading bacteria compared to control mice exposed to the toxin alone. These results suggest a potential novel therapeutic approach that can be developed for MC-LR induced toxicity