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Biomedical potential of marine cyanobacteria and bacteria associated with benthic invertebrates from Singapore
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Type
Thesis
Author
Gupta, Deepak Kumar
Supervisor
Tan, Lik Tong
Abstract
This thesis describes the biological screening and chemical investigations of filamentous marine cyanobacteria and bacteria associated with benthic invertebrates, such as sponges and soft corals, collected at various marine habitats in Singapore. The various chapters presented in this thesis include topics on the biological screening of marine samples, isolation, structural elucidation, and stereochemical determination of natural products, as well as phylogenetic analysis of isolated marine bacteria. The thesis begins with chapter one, by providing a general introduction and importance of marine natural products in drug discovery and development efforts. Various potent compounds isolated from marine organisms that are currently used as leads for drug development are discussed in this chapter. In addition, the importance of marine cyanobacterial compounds and molecules derived from marine invertebrate microbes as potential drug source is being presented in this introductory chapter.
Chapter 2 describes the field survey of marine benthic cyanobacteria from Singapore waters. The initial investigation revealed the vast occurrence of these microbes in different intertidal areas, including Pulau Hantu Besar, Pulau Hantu Central Lagoon, Pulau Hantu Kechil, St. Johns Island, Pulau Subar Laut, Pulau Belakang Mati, and Pulau Seringat Kias. Based on morphological features, these marine cyanobacteria are members of the Lyngbya and Trichodesmium genera. Preliminary biological screenings were carried out on crude organic extracts prepared from cyanobacterial samples. Fractionations and HPLC isolation of an extract derived from samples of Lyngbya majuscula collected from Pulau Hantu yielded known lipopeptides, majusculamides A and B and cyclic depsipeptides, hantupeptins, and trungapeptins. The planar structures of these natural products were confirmed by using an array of 1D and 2D NMR spectroscopic analysis as well as comparisons of spectral data with known compounds. The results of this pilot study highlighted the bioactive potential of filamentous marine cyanobacteria as a source of marine natural products.
Chapter three details the isolation of know and new aplysiatoxin-related compounds from the marine cyanobacterium, Trichodesmium erythraeum (TLT/PSK/001), obtained from Pulau Seringat, Singapore. The structures of new analogs were elucidated by interpretation of extensive 1D and 2D NMR spectral data as well as comparisons of spectral data with known aplysiatoxins. Moreover, the antichikungunya activity of these aplysiatoxin-related compounds were revealed in this study for the first time.
Chapter four outlined an alternative analytical method for the absolute stereochemical determination of β-hydroxy acid units present in hantupeptins and trungapeptins. In this method, standard stereoisomers of the 3-hydroxy-2-methyloctanoic acid (Hmoa) moieties were initially synthesized using Evan’s auxiliary and Mitsunobu reaction. Mosher’s derivatization coupled with RP-HPLC analysis were then used to determine the absolute stereochemistry of the Hmoa unit in hantupeptin C and trungapeptin C.
As part of a drug discovery program from a marine microorganism, the biomedical potential of bacteria associated with common marine invertebrates found at Kusu and Semakau Islands carried out in the chapters five and six. These marine invertebrates include three sponge species, Geolloides fibulata, Clathria reinwardti, and Xestospongia testudinaria as well as three soft coral species Sacrophyton tenuispiculatum, Sinularia sp., and. Lobophytum crassum. Using a culture-dependent method, a total of 57 bacteria were isolated, and their crude organic extract was screened for biological activities in the brine shrimp toxicity assay, cytotoxicity (MOLT-4 cells line) and antibacterial assays.
The antibacterial activity was performed in two phases’ i.e. primary screening against E. coli and B. cereus and secondary screening against methicillin-resistant S. aureus (MRSA), E. faecalis V583, E. coli EC 598, P. aeruginosa, B. cenocepacia, and A. baumannii.
The significant biological activity of isolated bacterial isolates encouraged us to carry out large-scale fermentation for chemical analysis (NMR and LC-MS) and isolation of bioactive constituents. Identification of selected bacterial isolate was conducted based on their 16S rRNA gene sequences. Overall 62% of bacterial isolates displayed activity ≥ 50% at 1000 ppm. In which, 16%, 39% and 25% of bacterial isolates exhibited activity in brine shrimp toxicity, cytotoxicity (MOLT-4), and antibacterial assays, respectively.
In chapter seven, we explored the expression of cryptic natural products through co-culturing of selected marine bacteria isolated from benthic invertebrates. A pilot study on micro-scale co-culturing of five selected soft coral-associated bacteria for induced metabolite's production was performed. We compared the 1H NMR spectra and LC-MS profile of co-culture extracts with the corresponding monoculture. As a result, two co-culture consortia exhibited different peaks when compared with the corresponding monocultures suggesting that co-culturing could induce the biosynthesis pathway of cryptic natural products. Finally, the thesis ends up with a concluding chapter eight summarizing the results presented in the preceding chapters as well as thoughts on future work.
Chapter 2 describes the field survey of marine benthic cyanobacteria from Singapore waters. The initial investigation revealed the vast occurrence of these microbes in different intertidal areas, including Pulau Hantu Besar, Pulau Hantu Central Lagoon, Pulau Hantu Kechil, St. Johns Island, Pulau Subar Laut, Pulau Belakang Mati, and Pulau Seringat Kias. Based on morphological features, these marine cyanobacteria are members of the Lyngbya and Trichodesmium genera. Preliminary biological screenings were carried out on crude organic extracts prepared from cyanobacterial samples. Fractionations and HPLC isolation of an extract derived from samples of Lyngbya majuscula collected from Pulau Hantu yielded known lipopeptides, majusculamides A and B and cyclic depsipeptides, hantupeptins, and trungapeptins. The planar structures of these natural products were confirmed by using an array of 1D and 2D NMR spectroscopic analysis as well as comparisons of spectral data with known compounds. The results of this pilot study highlighted the bioactive potential of filamentous marine cyanobacteria as a source of marine natural products.
Chapter three details the isolation of know and new aplysiatoxin-related compounds from the marine cyanobacterium, Trichodesmium erythraeum (TLT/PSK/001), obtained from Pulau Seringat, Singapore. The structures of new analogs were elucidated by interpretation of extensive 1D and 2D NMR spectral data as well as comparisons of spectral data with known aplysiatoxins. Moreover, the antichikungunya activity of these aplysiatoxin-related compounds were revealed in this study for the first time.
Chapter four outlined an alternative analytical method for the absolute stereochemical determination of β-hydroxy acid units present in hantupeptins and trungapeptins. In this method, standard stereoisomers of the 3-hydroxy-2-methyloctanoic acid (Hmoa) moieties were initially synthesized using Evan’s auxiliary and Mitsunobu reaction. Mosher’s derivatization coupled with RP-HPLC analysis were then used to determine the absolute stereochemistry of the Hmoa unit in hantupeptin C and trungapeptin C.
As part of a drug discovery program from a marine microorganism, the biomedical potential of bacteria associated with common marine invertebrates found at Kusu and Semakau Islands carried out in the chapters five and six. These marine invertebrates include three sponge species, Geolloides fibulata, Clathria reinwardti, and Xestospongia testudinaria as well as three soft coral species Sacrophyton tenuispiculatum, Sinularia sp., and. Lobophytum crassum. Using a culture-dependent method, a total of 57 bacteria were isolated, and their crude organic extract was screened for biological activities in the brine shrimp toxicity assay, cytotoxicity (MOLT-4 cells line) and antibacterial assays.
The antibacterial activity was performed in two phases’ i.e. primary screening against E. coli and B. cereus and secondary screening against methicillin-resistant S. aureus (MRSA), E. faecalis V583, E. coli EC 598, P. aeruginosa, B. cenocepacia, and A. baumannii.
The significant biological activity of isolated bacterial isolates encouraged us to carry out large-scale fermentation for chemical analysis (NMR and LC-MS) and isolation of bioactive constituents. Identification of selected bacterial isolate was conducted based on their 16S rRNA gene sequences. Overall 62% of bacterial isolates displayed activity ≥ 50% at 1000 ppm. In which, 16%, 39% and 25% of bacterial isolates exhibited activity in brine shrimp toxicity, cytotoxicity (MOLT-4), and antibacterial assays, respectively.
In chapter seven, we explored the expression of cryptic natural products through co-culturing of selected marine bacteria isolated from benthic invertebrates. A pilot study on micro-scale co-culturing of five selected soft coral-associated bacteria for induced metabolite's production was performed. We compared the 1H NMR spectra and LC-MS profile of co-culture extracts with the corresponding monoculture. As a result, two co-culture consortia exhibited different peaks when compared with the corresponding monocultures suggesting that co-culturing could induce the biosynthesis pathway of cryptic natural products. Finally, the thesis ends up with a concluding chapter eight summarizing the results presented in the preceding chapters as well as thoughts on future work.
Date Issued
2016
Call Number
RS160.7 Dee
Date Submitted
2016