Investigations on Marine Natural Products from Indo-Pacific Nudibranchia (Mollusca: Gastropoda): Chemoecology, Medicinal Potential & Toxin Resistance
Investigations on Marine Natural Products from Indo-Pacific Nudibranchia (Mollusca: Gastropoda): Chemoecology, Medicinal Potential & Toxin Resistance
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DissertationDate of Exam
11.03.2022Date of Publication
22.03.2022Advisor
König, Gabriele M.Co-Referee
Wägele, HeikeMetadata
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Abstract
This thesis focusses on the investigation of marine natural products (MNPs) from nudibranchs of the Central Indo-Pacific Ocean and their food sources. MNPs are important lead structures in the drug discovery process, as they can provide important chemical scaffolds with advantageous pharmacological properties. The exceptional biodiversity, intense competition and feeding pressure in coral reefs of the Central Indo‑Pacific Ocean led to a vast chemical diversity and variety of bioactive MNPs, especially in exposed, sessile, and slow-moving organisms. Sea slugs of the order Nudibranchia have completely lost their protective shell. To protect themselves from predation, several defence mechanisms have evolved, including cryptic or aposematic coloration and mimicry, and the ability to steal, incorporate and use the defence system of their prey, such as MNPs, which can be used for chemical defence. Here we show that one of the largest species, Phyllodesmium longicirrum (Nudibranchia: Aeolidioidea: Myrrhinidae), acquires and accumulates terpenoid compounds from their prey, especially cembranoid diterpenes. Investigation of the lipophilic extract of a single P. longicirrum specimen led to the isolation of nine terpenoid metabolites: the cembranoid diterpene (2R,11R,12R)-isosarcophytoxide (1), its epimer (2S,11R,12R)-isosarcophytoxide (2), the related (3R,4S,11R,12R)-bisepoxide (3), and the γ-lactone bearing cembranes sarcophytonin B (4) and 13-dehydroxysarcoglaucol-16-one (5); the two polycyclic diterpenes 4-oxo-chatancin (6) and 1-O-methyl-4-oxo-chatancin (7); and the two biscembranes bisglaucumlide L (8) and bisglaucumlide M (9). P. longicirrum sequestered these metabolites from its prey, most likely alcyonacean soft corals of the genus Sarcophyton. Metabolites (6) and (2) significantly deterred feeding by the generalist pufferfish Canthigaster solandri, emphasizing an ecological role in defence for at least some of these terpenoids. Interestingly, compound (1), the epimer of compound (2), showed no significant deterrent activity, which suggests that feeding deterrence might be conformation-dependant. The shift from hydrozoan to alcyonacean food sources and the successful scavenging of this prey likely enhanced radiation and speciation in the genus Phyllodesmium. Sequestration of photosynthetic zooxanthellae and defensive metabolites provides additional nutrition and a metabolically cheap and effective protection, which can lead to a higher viability. The structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin was isolated from Doriprismatica stellata (Nudibranchia: Doridina: Chromodorididae) nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina (Porifera: Demospongiae: Dictyoceratida). The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32). Structural elucidation revealed that the cyclopropane ring had an unprecedented position annelated to ring A and, unlike previously reported scalaranes, it bears two acetoxy groups at C-11 and C-24 of ring C, instead of being functionalized at C-12. The occurrence of this scalarane in all three samples further supports the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. Furthermore, the presence of this molecule in the egg ribbons of D. stellata suggests that the nudibranch passes the scalarane on to its offspring, most likely for protective purposes. Chromodoris sea slugs, a genus of aposematically colorful nudibranchs (Nudibranchia: Doridina: Chromodorididae), live and feed on noxious sponges, from which they sequester deterrent and toxic molecules. Here we show that five closely related species of Chromodoris sea slugs (C. annae, C. dianae, C. lochi, C. strigata and C. willani) selectively accumulate the cytotoxin LatA from their food source Cacospongia mycofijiensis (Porifera: Demospongiae: Dictyoceratida). The distribution of LatA was visualized for the first time within the body of two Chromodoris species using MALDI MS‑Imaging, showing that LatA was accumulated and stored specifically throughout the mantle tissue, mucus glands, and especially in vacuoles of the mantle dermal formations (MDFs). These results emphasize the importance of MDFs as repository for the storage of highly concentrated amounts of toxic molecules, in this case LatA. Furthermore, LatA was secreted along with the mucus and mucus trail, where it possibly serves as a semiochemical in short-range chemical communication, deterring predators and attracting mating partners. Using HEK-293 cells and fluorescence microscopy, we show that the isolated LatA is bioactive. Its cytotoxicity results from binding to G-actin monomers, one of the most essential eukaryotic proteins, which prohibits their polymerization to F-actin and additionally severs the F-actin network, which leads to the collapse of the cytoskeleton and death of the cell. In vivo toxicity experiments with direct administration of LatA showed 100% mortality in E. viridis, but 0% mortality in Chromodoris heterobranchs. Investigation of an underlying molecular resistance mechanism against the cytotoxic activity of LatA led us to amplify, sequence, examine and compare heterobranch actin genes, revealing a novel actin isoform in all investigated Chromodoris species. This isoactin carries two crucial amino acid substitutions, D187G and R206T, at the the ‘nucleotide binding’ cleft, the binding site of LatA. These substitutions likely lead to target-site modifications, interfering with LatA binding, hence, causing LatA insensitivity. Isoactin D187G/R206T is suggested to be a prerequisite for Chromodoris nudibranchs to sequester latrunculins from Negombata magnifica and Cacospongia mycofijiensis sponges. It would allow Chromodoris sea slugs to store and utilize LatA for their own defence, without having to suffer from its cytotoxicity.
Subjects
Chemoökologie, Naturstoffe, Nudibranchia, Resistenz, Sekundärmetabolit, Chemoecology, Natural products, Resistance, Secondary metabolites
Classification (DDC)
570 Biowissenschaften, BiologieHertzer, Cora: Investigations on Marine Natural Products from Indo-Pacific Nudibranchia (Mollusca: Gastropoda): Chemoecology, Medicinal Potential & Toxin Resistance. - Bonn, 2022. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-66023
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-66023
@phdthesis{handle:20.500.11811/9700,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-66023,
author = {{Cora Hertzer}},
title = {Investigations on Marine Natural Products from Indo-Pacific Nudibranchia (Mollusca: Gastropoda): Chemoecology, Medicinal Potential & Toxin Resistance},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2022,
month = mar,
note = {This thesis focusses on the investigation of marine natural products (MNPs) from nudibranchs of the Central Indo-Pacific Ocean and their food sources. MNPs are important lead structures in the drug discovery process, as they can provide important chemical scaffolds with advantageous pharmacological properties. The exceptional biodiversity, intense competition and feeding pressure in coral reefs of the Central Indo‑Pacific Ocean led to a vast chemical diversity and variety of bioactive MNPs, especially in exposed, sessile, and slow-moving organisms. Sea slugs of the order Nudibranchia have completely lost their protective shell. To protect themselves from predation, several defence mechanisms have evolved, including cryptic or aposematic coloration and mimicry, and the ability to steal, incorporate and use the defence system of their prey, such as MNPs, which can be used for chemical defence. Here we show that one of the largest species, Phyllodesmium longicirrum (Nudibranchia: Aeolidioidea: Myrrhinidae), acquires and accumulates terpenoid compounds from their prey, especially cembranoid diterpenes. Investigation of the lipophilic extract of a single P. longicirrum specimen led to the isolation of nine terpenoid metabolites: the cembranoid diterpene (2R,11R,12R)-isosarcophytoxide (1), its epimer (2S,11R,12R)-isosarcophytoxide (2), the related (3R,4S,11R,12R)-bisepoxide (3), and the γ-lactone bearing cembranes sarcophytonin B (4) and 13-dehydroxysarcoglaucol-16-one (5); the two polycyclic diterpenes 4-oxo-chatancin (6) and 1-O-methyl-4-oxo-chatancin (7); and the two biscembranes bisglaucumlide L (8) and bisglaucumlide M (9). P. longicirrum sequestered these metabolites from its prey, most likely alcyonacean soft corals of the genus Sarcophyton. Metabolites (6) and (2) significantly deterred feeding by the generalist pufferfish Canthigaster solandri, emphasizing an ecological role in defence for at least some of these terpenoids. Interestingly, compound (1), the epimer of compound (2), showed no significant deterrent activity, which suggests that feeding deterrence might be conformation-dependant. The shift from hydrozoan to alcyonacean food sources and the successful scavenging of this prey likely enhanced radiation and speciation in the genus Phyllodesmium. Sequestration of photosynthetic zooxanthellae and defensive metabolites provides additional nutrition and a metabolically cheap and effective protection, which can lead to a higher viability. The structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin was isolated from Doriprismatica stellata (Nudibranchia: Doridina: Chromodorididae) nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina (Porifera: Demospongiae: Dictyoceratida). The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32). Structural elucidation revealed that the cyclopropane ring had an unprecedented position annelated to ring A and, unlike previously reported scalaranes, it bears two acetoxy groups at C-11 and C-24 of ring C, instead of being functionalized at C-12. The occurrence of this scalarane in all three samples further supports the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. Furthermore, the presence of this molecule in the egg ribbons of D. stellata suggests that the nudibranch passes the scalarane on to its offspring, most likely for protective purposes. Chromodoris sea slugs, a genus of aposematically colorful nudibranchs (Nudibranchia: Doridina: Chromodorididae), live and feed on noxious sponges, from which they sequester deterrent and toxic molecules. Here we show that five closely related species of Chromodoris sea slugs (C. annae, C. dianae, C. lochi, C. strigata and C. willani) selectively accumulate the cytotoxin LatA from their food source Cacospongia mycofijiensis (Porifera: Demospongiae: Dictyoceratida). The distribution of LatA was visualized for the first time within the body of two Chromodoris species using MALDI MS‑Imaging, showing that LatA was accumulated and stored specifically throughout the mantle tissue, mucus glands, and especially in vacuoles of the mantle dermal formations (MDFs). These results emphasize the importance of MDFs as repository for the storage of highly concentrated amounts of toxic molecules, in this case LatA. Furthermore, LatA was secreted along with the mucus and mucus trail, where it possibly serves as a semiochemical in short-range chemical communication, deterring predators and attracting mating partners. Using HEK-293 cells and fluorescence microscopy, we show that the isolated LatA is bioactive. Its cytotoxicity results from binding to G-actin monomers, one of the most essential eukaryotic proteins, which prohibits their polymerization to F-actin and additionally severs the F-actin network, which leads to the collapse of the cytoskeleton and death of the cell. In vivo toxicity experiments with direct administration of LatA showed 100% mortality in E. viridis, but 0% mortality in Chromodoris heterobranchs. Investigation of an underlying molecular resistance mechanism against the cytotoxic activity of LatA led us to amplify, sequence, examine and compare heterobranch actin genes, revealing a novel actin isoform in all investigated Chromodoris species. This isoactin carries two crucial amino acid substitutions, D187G and R206T, at the the ‘nucleotide binding’ cleft, the binding site of LatA. These substitutions likely lead to target-site modifications, interfering with LatA binding, hence, causing LatA insensitivity. Isoactin D187G/R206T is suggested to be a prerequisite for Chromodoris nudibranchs to sequester latrunculins from Negombata magnifica and Cacospongia mycofijiensis sponges. It would allow Chromodoris sea slugs to store and utilize LatA for their own defence, without having to suffer from its cytotoxicity.},
url = {https://hdl.handle.net/20.500.11811/9700}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-66023,
author = {{Cora Hertzer}},
title = {Investigations on Marine Natural Products from Indo-Pacific Nudibranchia (Mollusca: Gastropoda): Chemoecology, Medicinal Potential & Toxin Resistance},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2022,
month = mar,
note = {This thesis focusses on the investigation of marine natural products (MNPs) from nudibranchs of the Central Indo-Pacific Ocean and their food sources. MNPs are important lead structures in the drug discovery process, as they can provide important chemical scaffolds with advantageous pharmacological properties. The exceptional biodiversity, intense competition and feeding pressure in coral reefs of the Central Indo‑Pacific Ocean led to a vast chemical diversity and variety of bioactive MNPs, especially in exposed, sessile, and slow-moving organisms. Sea slugs of the order Nudibranchia have completely lost their protective shell. To protect themselves from predation, several defence mechanisms have evolved, including cryptic or aposematic coloration and mimicry, and the ability to steal, incorporate and use the defence system of their prey, such as MNPs, which can be used for chemical defence. Here we show that one of the largest species, Phyllodesmium longicirrum (Nudibranchia: Aeolidioidea: Myrrhinidae), acquires and accumulates terpenoid compounds from their prey, especially cembranoid diterpenes. Investigation of the lipophilic extract of a single P. longicirrum specimen led to the isolation of nine terpenoid metabolites: the cembranoid diterpene (2R,11R,12R)-isosarcophytoxide (1), its epimer (2S,11R,12R)-isosarcophytoxide (2), the related (3R,4S,11R,12R)-bisepoxide (3), and the γ-lactone bearing cembranes sarcophytonin B (4) and 13-dehydroxysarcoglaucol-16-one (5); the two polycyclic diterpenes 4-oxo-chatancin (6) and 1-O-methyl-4-oxo-chatancin (7); and the two biscembranes bisglaucumlide L (8) and bisglaucumlide M (9). P. longicirrum sequestered these metabolites from its prey, most likely alcyonacean soft corals of the genus Sarcophyton. Metabolites (6) and (2) significantly deterred feeding by the generalist pufferfish Canthigaster solandri, emphasizing an ecological role in defence for at least some of these terpenoids. Interestingly, compound (1), the epimer of compound (2), showed no significant deterrent activity, which suggests that feeding deterrence might be conformation-dependant. The shift from hydrozoan to alcyonacean food sources and the successful scavenging of this prey likely enhanced radiation and speciation in the genus Phyllodesmium. Sequestration of photosynthetic zooxanthellae and defensive metabolites provides additional nutrition and a metabolically cheap and effective protection, which can lead to a higher viability. The structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin was isolated from Doriprismatica stellata (Nudibranchia: Doridina: Chromodorididae) nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina (Porifera: Demospongiae: Dictyoceratida). The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32). Structural elucidation revealed that the cyclopropane ring had an unprecedented position annelated to ring A and, unlike previously reported scalaranes, it bears two acetoxy groups at C-11 and C-24 of ring C, instead of being functionalized at C-12. The occurrence of this scalarane in all three samples further supports the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. Furthermore, the presence of this molecule in the egg ribbons of D. stellata suggests that the nudibranch passes the scalarane on to its offspring, most likely for protective purposes. Chromodoris sea slugs, a genus of aposematically colorful nudibranchs (Nudibranchia: Doridina: Chromodorididae), live and feed on noxious sponges, from which they sequester deterrent and toxic molecules. Here we show that five closely related species of Chromodoris sea slugs (C. annae, C. dianae, C. lochi, C. strigata and C. willani) selectively accumulate the cytotoxin LatA from their food source Cacospongia mycofijiensis (Porifera: Demospongiae: Dictyoceratida). The distribution of LatA was visualized for the first time within the body of two Chromodoris species using MALDI MS‑Imaging, showing that LatA was accumulated and stored specifically throughout the mantle tissue, mucus glands, and especially in vacuoles of the mantle dermal formations (MDFs). These results emphasize the importance of MDFs as repository for the storage of highly concentrated amounts of toxic molecules, in this case LatA. Furthermore, LatA was secreted along with the mucus and mucus trail, where it possibly serves as a semiochemical in short-range chemical communication, deterring predators and attracting mating partners. Using HEK-293 cells and fluorescence microscopy, we show that the isolated LatA is bioactive. Its cytotoxicity results from binding to G-actin monomers, one of the most essential eukaryotic proteins, which prohibits their polymerization to F-actin and additionally severs the F-actin network, which leads to the collapse of the cytoskeleton and death of the cell. In vivo toxicity experiments with direct administration of LatA showed 100% mortality in E. viridis, but 0% mortality in Chromodoris heterobranchs. Investigation of an underlying molecular resistance mechanism against the cytotoxic activity of LatA led us to amplify, sequence, examine and compare heterobranch actin genes, revealing a novel actin isoform in all investigated Chromodoris species. This isoactin carries two crucial amino acid substitutions, D187G and R206T, at the the ‘nucleotide binding’ cleft, the binding site of LatA. These substitutions likely lead to target-site modifications, interfering with LatA binding, hence, causing LatA insensitivity. Isoactin D187G/R206T is suggested to be a prerequisite for Chromodoris nudibranchs to sequester latrunculins from Negombata magnifica and Cacospongia mycofijiensis sponges. It would allow Chromodoris sea slugs to store and utilize LatA for their own defence, without having to suffer from its cytotoxicity.},
url = {https://hdl.handle.net/20.500.11811/9700}
}
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