A novel bacterial symbiont in the seaweed Gracilaria tenuistipitata and its promising anti-inflammatory and antiproliferative potential in relation to special secreted metabolites

Abstract

Marine macroalgae host compositionally complex microbiomes that constitute an evolutionarily refined, yet largely unexplored source of structurally diverse natural products. Thus, the bioactivities of the algae may be related to those of their associated microorganisms. Here we describe the discovery of strain S.100, an endophytic bacterium isolated from the red alga Gracilaria tenuistipitata and displaying remarkable anti-inflammatory and antiproliferative activities, through a primary screening on ethyl acetate (EtAc), chloroform and n-hexane extracts of its culture. S.100 was taxonomically identified to the species level by morphology observation, and sequence analyses of 16S rRNA and housekeeping rpoB genes. Its anti-inflammatory and antiproliferative activities, as well as toxicity, were further investigated by using standard procedures performed on the EtAc extract. This extract was also fractionated to five fractions and the fraction with the most pronounced activities was subject to liquid chromatography–high-resolution mass spectrometry (LC-HRMS) to identify its chemical components. The results show that S.100 belongs to a Bacillus species reported for the first time to be associated with Gracilaria tenuistipitata . The EtAc extract of its culture selectively inhibited proliferation of human cervical (HeLa, IC 50 = 31.87 μg/mL) and hepatocellular (HepG2, IC 50 = 63.8 μg/mL) carcinoma cell lines, with substantially lower cytotoxicity toward normal human embryonic kidney cells (HEK 293T, IC 50 = 181.1 μg/mL). At 120 μg/mL, the extract also suppressed lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages. Toxicological assessment in zebrafish embryos revealed no developmental abnormalities below 200 mg/L and an LC50 of 421.6 mg/L at 96 h, consistent with a favorable safety profile. Bioactivity-guided fractionation, coupled with LC-HRMS analysis, putatively annotated 16 specialized metabolites in the most active fraction (fraction 4), including noteworthily bioactive cyclic lipopeptides (surfactins B and C), β-carboline and quinoline alkaloids (norharmane, harmane, and quinoline-4-carboxylic acid), and phenolic acids (gallic and 2,3-dihydroxybenzoic acids). Together, these findings show exciting biopharmaceutical application potential of S.100 and highlight macroalgal endophytes as a promising chemical source for the discovery of marine-derived therapeutics.