Microbial symbionts of marine invertebrates: opportunities for microbial biotechnology

Marine invertebrates are sources of a diverse array of bioactive metabolites with great potential for development as drugs and research tools. In many cases, microorganisms are known or suspected to be the biosynthetic source of marine invertebrate natural products. The application of molecular microbiology to the study of these relationships will contribute to basic biological knowledge and facilitate biotechnological development of these valuable resources. The bryostatin-producing bryozoan B. neritina and its specific symbiont "Candidatus Endobugula sertula" constitute one promising model system. Another fertile subject for investigation is the listhistid sponges that contain numerous bioactive metabolites, some of which originate from bacterial symbionts.     

Symbiotic and dietary marine microalgae as a source of bioactive molecules–experience from natural products research

Cyanobacterial metabolites have proven to be invaluable as tools in thedissection of signal transduction pathways in mammalian cells and some arecurrently under clinical evaluation as drug candidates. It is now also realizedthat cyanobacteria are the true biosynthetic origin of many bioactive moleculesisolated from marine invertebrates; marine invertebrates may sequestercyanobacteria through diet or by symbiosis. This review discusses thedietary-derived cyanobacterial origin of the dolastatins, potent cytotoxiccompounds, originally isolated from the Indian Ocean sea hare,Dolabella auricularia. A discussion on the dietarydissemination of cyanobacterial metabolites through the marine food chain isalso presented. Reference to the metabolites isolated fromDysidea sponges is given to illustrate their origin fromsymbiotic cyanobacteria associated with this organism.     

Phytoplankton trigger the production of cryptic metabolites in the marine actinobacterium Salinispora tropica

Filamentous members of the phylum Actinobacteria are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacterium Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide but half of its putative BGCs are still orphan. Although previous studies have used marine heterotrophs to induce orphan BGCs in Salinispora, its co-culture with marine phototrophs has yet to be investigated. Following the observation of an antimicrobial activity against a range of phytoplankton by S. tropica, we here report that the photosynthate released by photosynthetic primary producers influences its biosynthetic capacities with production of cryptic molecules and the activation of orphan BGCs. Our work, using an approach combining metabolomics and proteomics, pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from marine actinobacteria.     

海洋共附生微生物天然产物生物合成基因研究进展

对海洋无脊椎动物天然产物的研究表明,很多种活性物质的真正生产者是与其共生或附生的未培养微生物.克隆这些未培养微生物中特定活性物质的生物合成基因,不仅为活性物质的来源提供遗传学证据,也使通过异源表达相关生物合成基因来大量获取目标化合物成为可能.本文综述了来源于海绵、海鞘、苔藓虫、深海管状蠕虫和深海沉积物中共附生微生物天然产物生物合成基因簇的研究进展.     

Non-methylene-interrupted fatty acids from marine invertebrates: Occurrence,characterization and biological properties

Marine organisms, in particular invertebrates, have proved to be a major source of unique fatty acid (FA) structures originating from unusual biosynthetic pathways. Among them, non-methylene-interrupted (NMI) FA occur in various molluscs in the wide ranges of concentrations (up to 20%), such as the most often encountered 20:2 Δ5,11, 20:2 Δ5,13, 22:2 Δ7,13 or 22:2 Δ7,15. Such NMI FA have also been reported from algae, echinoderms, sponges, tropical rays, and many other invertebrates. The most intriguing marine invertebrates seem to be sponges that commonly contain very long-chain Δ5,9 FA. A third double bond can occur in the NMI FA as reported in some marine organisms, such as 20:3 Δ7,13,16 or 30:3 Δ5,9,23. Lipids of invertebrates from deep-sea hydrothermal and cold-seep vents gave rise to an intense research activity including reports on unprecedented NMI polyunsaturated FA. The bivalve molluscs are able to synthesize de novo the NMI FA but their precise biological interest is presently not well-known, although structural and functional roles in biological membranes have been suggested, in particular a higher resistance to oxidative processes and microbial lipases. Biosynthetic pathways of Δ5,9 FA in sponges were demonstrated up to C₂₆ FA structures and include particular elongation and desaturation steps. Recently, intense research effort has been conducted to investigate the biomedical potential of these unusual FA. Thus, Δ5,9 FA displayed interesting antiplasmodial activity. The most promising FA topoisomerase I inhibitors to date seem to be the long-chain Δ5,9 FA. This inhibitory activity is probably partially responsible for the toxicity displayed by some of the Δ5,9 FA towards cancer cell lines.     

Carbohydrate-binding proteins of marine invertebrates

The information on the carbohydrate specificity and molecular organization of some carbohydrate-binding proteins (lectins) of marine invertebrates is reported. Antiviral activity of some of the lectins against human immunodeficiency virus has been studied. Lectins of marine invertebrates are promising tools for studying natural glycoconjugates and cell effectors in vitro.     

Mining of Egypt’s Red Sea invertebrates for potential bioactive producers

Objective

The objective of this work was to isolate bacteria from Red Sea invertebrates, determine their antimicrobial activity, and screen for the biosynthetic gene clusters [polyketides (PKs) and nonribosomal peptides (NRPs)] which could be involved in the production of bioactive secondary metabolites.

Result

Eleven different samples of marine invertebrates’ were collected from Egypt’s Red Sea (El-Tor-Sharm El-Sheikh and Hurghada) by scuba diving, and a total 80 isolates of the associated microorganisms were obtained from the cultivation on six different cultural medium. Seven isolates of them showed an antimicrobial activity against five pathogenic reference strains, while the most active antimicrobial agent was isolate number HFF-8 which was 99% identical to Bacillus amyloliquefaciens. HFF-8’s extract showed positive results against Gram negative bacteria, Gram positive bacteria and yeast. Moreover, the isolates gave positive bands when screened for the presence of PK synthase (PKS) I and II and NRP synthetase (NRPS) I and II biosynthetic genes, those biosynthetic fragments when cloned and sequenced were primitively predicted as biosynthetic fragments for kirromycin and leinamycin production by NaPDoS program with 56 and 55%, respectively.

Conclusion

The Red Sea can provide a sustainable solution to combat bacterial resistance. The contribution of this work is that B. amyloliquefaciens was isolated from Heteroxenia fuscescens, Red Sea, Egypt. Moreover, the bacterial extract showed a broad spectrum with a potent antimicrobial activity.

     

Carbohydrate-binding proteins of marine invertebrates

The information on the carbohydrate specificity and molecular organization of some carbohydrate-binding proteins (lectins) of marine invertebrates is reported. Antiviral activity of some of the lectins against human immunodeficiency virus has been studied. Lectins of marine invertebrates are promising tools for studying natural glycoconjugates and cell effectors in vitro.     

Are there adequate data to assess how well theories of rarity apply to marine invertebrates?

Understanding the biology of rare species is a very important part of conservation biology. Most of our current understanding of rarity has, however, come from studies of terrestrial plants, birds, mammals and some insects. Freshwater and marine habitats are underrepresented in published studies of rare species or conservation biology. We therefore have little knowledge about how well our understanding of what makes particular species rare and how rare species persist applies to marine invertebrates which form a major component of coastal biodiversity. In this review, I examine some theories about rarity with respect to intertidal and shallow subtidal invertebrates to identify whether there are adequate data to apply these theories to marine invertebrates and how well such theories apply. The general conclusions are that the lack of quantitative data on abundances, ranges, habitat-requirements, dispersal and connectedness among populations for marine invertebrates means that their status as rare species cannot really be assessed appropriately. It is also unlikely that, without extensive sampling programmes and considerable expense, adequate data could be obtained for these small, cryptic animals, which typically have very patchy, variable and unpredictable patterns of distribution and abundance. Intertidal and subtidal assemblages are diverse, including species with many different life-histories from many phyla, occupying the same suite of habitats. It is therefore suggested that future research on rare organisms in marine habitats should build upon the long and successful history of experimental marine studies to test specific hypotheses about processes influencing rarity in the field. Such studies would not only add a new dimension to our current understanding of rarity, but would also provide badly-needed data on the status of rare marine invertebrates. abundances, invertebrates, marine, range, rarity     

Natural products from marine invertebrates against <Emphasis Type="Italic">Leishmania</Emphasis> parasites: a comprehensive review

Parasitic infections by Leishmania parasites remains a severe public health problem, especially in developing countries where it is highly endemic. Chemotherapy still remains a first option for the treatment of those diseases, despite the fact that available drugs exhibit a variety of shortcomings. Thus, innovative, less toxic more affordable and effective antileishmanial agents are urgently needed. The marine environment holds an immeasurable bio- and chemical diversity, being a valuable source of natural products with therapeutic potential. As invertebrates comprise about 60 % of all marine organisms, bioprospecting this class of organisms for antileishmanial properties may unravel unique and selective hit molecules. In this context, this review covers results on the literature of marine invertebrate extracts and pure compounds evaluated against Leishmania parasites mainly by in vitro methods. It comprises results obtained from the phyla Porifera, Cnidaria, Bryozoa (Ectoprota), Mollusca, Echinodermata, Annelida, Cetnophora, Platyhelminthes, sub phyla Crustacea (phylum Arthropoda) and Tunicata (phylum Chordata). Moreover, structure–activity relationships and possible mechanisms of action are mentioned, whenever available information is provided. About 70 species of marine invertebrates belonging to seven different phyla are included in this work. Besides a variety of crude extracts, a total of 140 pure compounds was tested against different Leishmania species. Although the research on the antileishmanial potential of marine invertebrates is in its early beginnings, promising results have been achieved that encourage further research. As more extracts and compounds are being screened, the possibility of finding active and selective antileishmanial molecules increases, rising new hope in the search for new treatments against leishmaniases.     

Highlights of marine invertebrate-derived biosynthetic products: their biomedical potential and possible production by microbial associants

Coral reefs are among the most productive marine ecosystems and are the source of a large group of structurally unique biosynthetic products. Annual reviews of marine natural products continue to illustrate that the most prolific source of bioactive compounds consist of coral reef invertebrates-sponges, ascidians, mollusks, and bryozoans. This account examines recent milestone developments pertaining to compounds from invertebrates designated as therapeutic leads for biomedical discovery. The focus is on the secondary metabolites, their inspirational structural scaffolds and the possible role of micro-organism associants in their biosynthesis. Also important are the increasing concerns regarding the collection of reef invertebrates for the discovery process. The case examples considered here will be useful to insure that future research to unearth bioactive invertebrate-derived compounds will be carried out in a sustainable and environmentally conscious fashion. Our account begins with some observations pertaining to the natural history of these organisms. Many still believe that a serious obstacle to the ultimate development of a marine natural product isolated from coral reef invertebrates is the problem of compound supply. Recent achievements through total synthesis can now be drawn on to forcefully cast this myth aside. The tools of semisynthesis of complex natural products or insights from SAR efforts to simplify an active pharmacophore are at hand and demand discussion. Equally exciting is the prospect that invertebrate-associated micro-organisms may represent the next frontier to accelerate the development of high priority therapeutic candidates. Currently in the United States there are two FDA approved marine-derived therapeutic drugs and two others that are often cited as being marine-inspired. This record will be examined first followed by an analysis of a dozen of our favorite examples of coral reef invertebrate natural products having therapeutic potential. The record of using complex scaffolds of marine invertebrate products as the starting point for development will be reviewed by considering eight case examples. The potential promise of developing invertebrate-derived micro-organisms as the starting point for further exploration of therapeutically relevant structures is considered. Also significant is the circumstance that there are some 14 sponge-derived compounds that are available to facilitate fundamental biological investigations.     

Distribution of opine dehydrogenases and lactate dehydrogenase activities in marine animals

• 1.1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence.
• 2.2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata.
• 3.3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated.
• 4.4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.

     

Antimicrobial peptides from marine invertebrates: challenges and perspectives in marine antimicrobial peptide discovery

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on the purification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptides are promising candidates, because their initial interaction with microbes is through binding to lipids. The interference with such a fundamental cell structure is assumed to hamper resistance development. In the present review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing the isolation and activity of these natural antibiotics. The marine environment is relatively poorly explored in terms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in close proximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobial peptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient and rapid inhibitors of microbial growth.     

Frequency of injury and the ecology of regeneration in marine benthic invertebrates

Many marine invertebrates are able to regenerate lost tissue following injury, but regeneration can come at a cost to individuals in terms of reproduction, behavior and physiological condition, and can have effects that reach beyond the individual to impact populations, communities, and ecosystems. For example, removal and subsequent regeneration of clams' siphons, polychaetes' segments, and brittlestars' arms can represent significant energetic input to higher trophic levels. In marine soft-sediment habitats, injury changes infaunal bioturbation rates and thus secondarily influences sediment-mediated competition, adult-larval interactions, and recruitment success. The importance of injury and regeneration as factors affecting the ecology of marine invertebrate communities depends on the frequency of injury, as well as on individual capacity for, and speed of, regeneration. A key question to answer is: "How frequently are marine benthic invertebrates injured?" Here, I review the sources and the frequencies of injury in a variety of marine invertebrates from different benthic habitats, discuss challenges, and approaches for accurately determining injury rates in the field, consider evidence for species-specific, temporal and geographic variation in injury rates, and present examples of indirect effects of injury on marine invertebrates to illustrate how injury and regeneration can modify larger-scale ecological patterns and processes.     

The cost of being a caring mother: the ignored factor in the reproduction of marine invertebrates

Investment in reproduction ranges from gamete production to active parental care, and marine invertebrates span this range. However, the cost of parental care has not yet been systematically quantified, nor incorporated into life history studies of marine invertebrates, in contrast to most other animal taxa. Since oxygen is a limiting factor in egg masses of marine invertebrates, we studied patterns of oxygen partial pressure over time in embryo masses of Brachyuran crabs, and correlated these results with the cost of providing oxygen to the embryos. We found that: (1) oxygen is limiting in the embryo masses, (2) female crabs show an active brooding behaviour that we think helps to provide oxygen to the embryo mass, and (3) there is a substantial parental investment associated with brooding behaviours. Oxygen limitation and parental investment seem to be associated in many taxa of marine invertebrates, and we suggest that oxygen provision to the embryos may be a critical factor determining parental investment in this group.     

Drug Discovery from Marine Microbes

The marine environment has been a source of more than 20,000 inspirational natural products discovered over the past 50 years. From these efforts, 9 approved drugs and 12 current clinical trial agents have been discovered, either as natural products or as molecules inspired from the natural product structure. To a significant degree, these have come from collections of marine invertebrates largely obtained from shallow-water tropical ecosystems. However, there is a growing recognition that marine invertebrates are oftentimes populated with enormous quantities of “associated” or symbiotic microorganisms and that microorganisms are the true metabolic sources of these most valuable of marine natural products. Also, because of the inherently multidisciplinary nature of this field, a high degree of innovation is characteristic of marine natural product drug discovery efforts.     

Overview of the chemical ecology of benthic marine invertebrates along the western Antarctic peninsula

Thirteen years ago in a review that appeared in the American Zoologist, we presented the first survey of the chemical and ecological bioactivity of Antarctic shallow-water marine invertebrates. In essence, we reported that despite theoretical predictions to the contrary the incidence of chemical defenses among sessile and sluggish Antarctic marine invertebrates was widespread. Since that time we and others have significantly expanded upon the base of knowledge of Antarctic marine invertebrates' chemical ecology, both from the perspective of examining marine invertebrates in new, distinct geographic provinces, as well as broadening the evaluation of the ecological significance of secondary metabolites. Importantly, many of these studies have been framed within established theoretical constructs, particularly the Optimal Defense Theory. In the present article, we review the current knowledge of chemical ecology of benthic marine invertebrates comprising communities along the Western Antarctic Peninsula (WAP), a region of Antarctica that is both physically and biologically distinct from the rest of the continent. Our overview indicates that, similar to other regions of Antarctica, anti-predator chemical defenses are widespread among species occurring along the WAP. In some groups, such as the sponges, the incidence of chemical defenses against predation is comparable to, or even slightly higher than, that found in tropical marine systems. While there is substantial knowledge of the chemical defenses of benthic marine invertebrates against predators, much less is known about chemical anti-foulants. The sole survey conducted to date suggests that secondary metabolites in benthic sponges are likely to be important in the prevention of fouling by benthic diatoms, yet generally lack activity against marine bacteria. Our understanding of the sensory ecology of Antarctic benthic marine invertebrates, despite its great potential, remains in its infancy. For example, along the WAP, community-level non-consumptive effects occur when amphipods chemically sense fish predators and respond by seeking refuge in chemically-defended macroalgae. Such interactions may be important in releasing amphipods from predation pressure and facilitating their unusually high abundances along the WAP. Moreover, recent studies on the sensory biology of the Antarctic keystone sea star Odontaster validus indicate that chemotactile-mediated interactions between conspecifics and other sympatric predatory sea stars may have significant ramifications in structuring community dynamics. Finally, from a global environmental perspective, understanding how chemical ecology structures marine benthic communities along the WAP must increasingly be viewed in the context of the dramatic impacts of rapid climatic change now occurring in this biogeographic region.     

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.     

Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products

Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culture-dependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both gram-positive and gram-negative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, and Vibrio mimicus.     

Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements

The establishment of cell lines from marine invertebrates has been encountered with obstacles. Contrary to insects and arachnids where the development of a variety of cell lines has become routine, there is no single established cell line from marine invertebrates. This review examines the activity in the field of marine invertebrate cell cultures within the last decade (1988–1998). During this period, attempts (90 peer reviewed studies in addition to many other abstracts, chapters in books, symposia presentations and reports) were limited to a few species within only six phyla (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, Urochordata; in addition to freshwater/terrestrial annelids and platyhelminths). These studies which are summarized here, on one hand indicated ubiquitous problems and on the other, unique characterizations to each phylum studied. Only one-third of the studies revealed cultures of 1 month or longer but most of these were long-term cultures found or suspiciously considered to be contaminated by other unicellular eukaryotic organisms, mainly by thraustochytrids. Three unique approaches/obstacles for marine invertebrate cell cultures (source of cell, cryopreservation and eukaryotic contaminants) are further discussed. The overall impact of recent improvements and developed protocols raises the suggestion for testing different, novel routes in the establishment of cell cultures from marine invertebrates.