Browsing by Author "Pheiffer, Fazlin"

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    Bioassay-guided fractionation leads to the detection of cholic acid generated by the rare thalassomonas sp.
    (Pubmed, 2023) Schneider, Yannik K.-H.; Pheiffer, Fazlin; Holst Hansen, Espen; Hammer Andersen, Jeanette
    Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment
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    Investigating the antimicrobial potential of Thalassomonas actiniarum
    (University of the Western Cape, 2020) Pheiffer, Fazlin; Trindade, Marla
    bioassay guided isolation approach was then used to isolate the high molecular weight antibacterial compound (50kDa-100kDa) from T. actiniarum fermentations. With common protein isolation, purification and detection methods failing to provide insight into the nature of the antibacterial compound, we hypothesized that the active agent is not proteinaceous in nature and may be a high molecular weight exopolysaccharide. Extraction and antibacterial screening of the exopolysaccharide fraction from T. actiniarum showed antibacterial activity as well as lytic activity when subjected to a zymography assay using Pseudomonas putida whole cells as a substrate. Additionally, the biosynthetic pathways for the production of poly-β-1, 6-N-acetyl-glucosamine (PNAG), an exopolysaccharide involved in biofilm formation and chondroitin sulfate, a known and industrially important glycosaminoglycan with antibacterial and anti-inflammatory activity was identified and the mechanism may be novel. Genome mining identified a variety of novel secondary metabolite gene clusters which could potentially encode other novel bioactivities. Therefore a bioassay guided isolation, focused on the small (<3kDa) molecules, was pursued. Secondary metabolites were extracted, fractionated and screened for biofilm inhibition, antibacterial and anticancer activity and activity was observed in all assays. Active fractions were dereplicated by UHPLC-QToF-MS and compounds of interest were isolated using mass guided preparative HPLC. The purity of the isolated compounds was assessed using UHPLC-QToF-MS and NMR and the structure of the target compounds elucidated. Structures that could be determined were the bile acids cholic acid and 3-oxo cholic acid and although not responsible for the observed activities, this is the first report of bile acid production for this genus. This is the first study investigating the bioactive potential of the strain and the first demonstrating that T. actiniarum is a promising source of potentially novel pharmaceutically relevant natural products depicted through both culture-dependent and culture-independent approaches.
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    Investigating the antimicrobial potential of Thalassomonas actiniarum
    (University of Western Cape, 2020) Pheiffer, Fazlin; Trindade, Marla; van Zyl, Leonardo
    The World Health Organisation predicts that by the year 2050, 10 million people could die annually as a result of infections caused by multidrug resistant bacteria. Individuals with compromised immune systems, caused by underlying disease such as HIV, MTB and COVID-19, are at a greater risk. Antibacterial resistance is a global concern that demands the discovery of novel drugs. Natural products, used since ancient times to treat diseases, are the most successful source of new drug candidates with bioactivities including antibiotic, antifungal, anticancer, antiviral, immunosuppressive, anti-inflammatory and biofilm inhibition. Marine bioprospecting has contributed significantly to the discovery of novel bioactive NPs with unique structures and biological activities, superior to that of compounds from terrestrial origin. Marine invertebrate symbionts are particularly promising sources of marine NPs as the competition between microorganisms associated with invertebrates for space and nutrients is the driving force behind the production of antibiotics, which also constitute pharmaceutically relevant natural products.