Bioprospecting of marine microorganisms for the discovery of antibacterial compounds - Isolation, structure elucidation and bioactivity assessment of marine microbial natural products

Abstract

<p>Infectious diseases have been a problem for humans since the beginning of human existence. The “golden age” of antibiotics started at the end of the 1920s, with the discovery of penicillin by Sir Alexander Fleming. This was followed by the discovery of several life-saving antibiotics. The number of new marketed antibiotics has declined, and most pharmaceutical companies are no longer working in antibiotic development. This in itself would be unproblematic, had it not been for the rapid ability of bacteria to become resistant towards previously debilitating agents. The need for new antibiotics is therefore eminent. Natural products have been important contributors for antibiotic drug discovery and development. Microorganisms have been a particularly proliferative source of antibiotics, providing us with among others the penicillins, aminoglycosides, tetracyclines and polymyxins. Most naturally derived pharmaceuticals, including antibiotics, originate from terrestrial organisms. This is mainly because the terrestrial environment historically has been easier to access compared to the marine environment below the intertidal zone. The marine environment is highly diverse, and there is still a huge biodiversity that is yet to be explored. In this project, Arctic and sub-Arctic marine bacteria and fungi were cultivated and studied for their production of natural products. The cultures were extracted and fractionated, and the fractions were tested for bioactivity, mainly focusing on antibacterial activity. Using bioactivity-guided isolation, compounds were isolated and structurally characterized. Finally, the bioactivity of the isolated compounds was broadly evaluated. <p>In paper I, a known siderophore, serratiochelin A, was isolated from a co-culture of two bacteria, <i>Serratia</i> sp. and <i>Shewanella</i> sp. The compound was not detected in axenic cultures, indicating that cocultivation triggered production. The acid-catalyzed degradation of serratiochelin A into serratiochelin C was also observed. Serratiochelin A had weak activity against <i>Staphylococcus aureus</i>, melanoma cells and non-malignant lung fibroblasts. No activity was observed for the degradation product serratiochelin C, indicating that the oxazoline moiety in the original compound is essential for the bioactivity. <p>In paper II, a marine bacterium <i>Lacinutrix</i> sp. was cultivated and studied for its ability to produce bioactive natural products. Through bioactivity-guided isolation, two new lyso-ornithine lipids were isolated, and their structures elucidated, showing that they only differed by the length of the hydrocarbon tail. Analysis by UHPLC-HR-MS indicate that the purified solutions are mixtures of isomers, but these were not possible to separate by preparative HPLC-MS. The compounds were evaluated for antibacterial activity and antiproliferative activity against human cells. Compound 1 displayed weak activity against <i>Streptococcus agalactiae</i>, while compound 2 had weak activity against melanoma cells. <p>In paper III, a new dimeric naphthopyrone substituted with a sulphate group was isolated in high yields from cultures of an obligate marine fungus in the family <i>Lulworthiaceae</i>. The compound was tested against an extended panel of clinical bacterial isolates and showed potent antibacterial activity against several clinical methicillin-resistant <i>Staphylococcus aureus</i> isolates, with MICs down to 1.56 μg/mL. Acid-catalyzed degradation was also observed. The compound also displayed moderate activities against three human cell lines: melanoma, hepatocellular carcinoma, and non-malignant lung fibroblast. <p>In paper IV, a new chlovalicin variant, chlovalicin B, was isolated from cultures of the obligate marine fungus <i>Digitatispora marina</i>. The fungus has previously been studied for its distribution in the marine environment but has not been extensively studied for its biosynthetic potential. The compound was isolated in low yields, and the structure was elucidated by NMR and HRMS experiments. The compound was assessed for a range of bioactivities and had weak antiproliferative activity against human melanoma cells.