Post by fritolay66 on Aug 1, 2008 1:41:35 GMT -5
I received some late night texts from a friend in So. Cali. I do not believe this avenue has been presented on this forum before. Here is the link and its contents.
ENDOPHYTES AND THEIR PROMISE FOR NEW MEDICINES AND PRODUCTS FOR AGRICULTURE AND INDUSTRY
Portions of the text taken from: Microbiology and Molecular Biology Reviews 67: 491-502. (2003). This information provides guidelines and rationale for the content of the film---“Jewels of the Jungle”
There is a general call for new antibiotics, chemotherapeutic agents and agrochemicals that are highly effective, possess low toxicity, and will have a minor environmental impact. This search is driven by the development of resistance in infectious microorganisms (e.g. Staphylococcus, Mycobacterium, Streptococcus) to existing compounds and by the menacing presence of naturally resistant organisms. The ingress to the human population of new diseases such as AIDS and SARS requires the discovery and development of new drugs to combat them. Not only do diseases such as AIDS require drugs that target them specifically, but so do new therapies for treating ancillary infections which are a consequence of a weakened immune system. Furthermore, others who are immunocompromised (e.g. cancer and organ transplant patients) are at risk by opportunistic pathogens, such as Aspergillus spp., Cryptococcus spp. and Candida spp., that normally are not major problems in the human population. In addition, more drugs are needed to efficiently treat parasitic protozoan and nematodal infections such as malaria, leshmaniasis, trypanomiasis and filairasis. Malaria alone is more effective in claiming lives each year than any other single infectious agent with the exception of the AIDS virus and Mycobacterium tuberculosis. Finally, because of safety and environmental problems, many synthetic agricultural agents have been, and currently are being targeted for removal from the market, which creates a need to find alternative ways to control farm pests and pathogens. Novel natural products and the organisms that make them offer opportunities for innovation in drug and agrochemical discovery. Exciting possibilities exist for those who are willing to venture into the wild and unexplored territories of the world to experience the excitement and thrill of engaging in the discovery of endophytes, their biology and potential usefulness.
NATURAL PRODUCTS AND TRADITIONAL APPROACHES IN MEDICINE
Natural products are naturally derived metabolites and/or byproducts from microorganisms, plants or animals. These products have been exploited for human use for thousands of years and plants have been the chief source of compounds used for medicine. Even today the largest users of traditional medicines are the Chinese with over 5000 plants, and plant products in their pharmacopoeia. In fact, the world’s best known and most universally used medicinal is aspirin (salicylic acid) which has it natural origins from the glycoside salicin which is found in many species of the plant genera Salix and Populus. Examples abound of natural product use, especially in small native populations in a myriad of remote locations on earth. For instance, certain tribal groups in the Amazon basin, the highland peoples of Papua New Guinea, and the aborigines of Australia each has identified certain plants to provide relief of symptoms varying from head colds to massive wounds and intestinal ailments. History also shows that now extinct civilizations had also discovered the benefits of medicinal plants. In fact, nearly 3000 years ago, the Mayans used fungi grown on roasted green corn to treat intestinal ailments. More recently, the Benedictine monks (800 AD) began to apply Papever somniferum as an anesthetic and pain reliever as the Greeks had done for years before. Many people, in past times, realized that leaf, root and stem concoctions had the potential to help them. These plant products, in general, enhanced the quality of life, reduced pain and suffering, and provided relief, even though an understanding of the chemical nature of bioactive compounds in these complex mixtures and how they functioned remained a mystery.
It was not until Pasteur discovered that fermentation is caused by living cells that people seriously began to investigate microbes as a source for bioactive natural products. Then, scientific serendipity and the power of observation provided the impetus to Fleming to usher in the antibiotic era via the discovery of penicillin from the fungus – Penicillium notatum. Since then, people have been engaged in the discovery and application of microbial metabolites with activity against both plant and human pathogens. Furthermore, the discovery of a plethora of microbes for applications that span a broad spectrum of utility in medicine (e.g. anticancer and immunosuppressant functions), agriculture and industry is now practical because of the development of novel, and sophisticated screening processes in both medicine and agriculture. These processes use individual organisms, cells, enzymes, and site directed techniques, many times in automated arrays, resulting in the rapid detection of promising leads for product development.
Even with untold centuries of human experience behind us and a movement into a modern era of chemistry and automation, it is still the case that natural product based compounds have had an immense impact on modern medicine since about 40% of prescription drugs are based on them. Furthermore, 49% of the new chemical products registered by the FDA are natural products or derivatives thereof. Excluding biologics, between 1989 and1995, 60% of approved drugs and pre-new drug application candidates were of natural origin. From 1983-1994, over 60% of all approved and pre-NDA stage cancer drugs were of natural origin as were 78% of all newly approved antibacterial agents. In fact, the world’s first billion dollar anticancer drug taxol is a natural product derived from the yew tree. Many other examples abound that illustrate the value and importance of natural products in modern civilizations.
Recently, however, natural product research efforts have lost popularity in many major drug companies and, in some cases, have been replaced entirely by combinatorial chemistry which is the automated synthesis of structurally related small molecules. In addition, many drug companies have developed interests in making products that have a larger potential profit base than antiinfectious drugs. These include compounds that provide social benefits, reduce the symptoms of allergies and arthritis, or ones that can sooth the stomach. It appears that this loss of interest can be attributed to the enormous effort and expense that is required to pick, and chose a biological source, then to isolate active natural products, decipher their structures, and begin the long road to product development. It is also apparent that combinatorial chemistry and other synthetic chemistries revolving around certain basic chemical structures is now serving as a never ending source of products to feed the screening robots of the drug industry. Within many large pharmaceutical companies, progress of professionals is primarily based upon numbers of compounds that can be produced and sent to the screening machines. This tends to work against the numerous steps needed even to find one compound in natural product discovery. It seems important to realize that the primary purpose of combinatorial chemistry should be to complement and assist the efforts of natural product drug discovery and development, not to supersede it. The natural product often serves as a lead molecule whose activity can be enhanced by manipulation through combinatorial and synthetic chemistry. Natural products have been the traditional pathfinder compounds with an untold diversity of chemical structures unparalleled by even the largest combinatorial databases.
ENDOPHYTES
It may also be true that a reduction in interest in natural products for use in drug development has happened as a result of people growing weary of dealing with the traditional sources of bioactive compounds including plants of the temperate zones, and microbes from a plethora of soil samples gathered indifferent parts of the world by armies of collectors. In other words, why do something different (working on endophytic microbes) when robots, combinatorial chemistry and molecular biology have arrived on the scene? Furthermore, the logic and rationale for time and effort spent on drug discovery using a target -site directed approach has been overwhelming.
While combinatorial synthesis produces compounds at random, secondary metabolites, defined as low molecular weight compounds not required for growth in pure culture, are produced as an adaptation for specific functions in nature. Shutz notes that certain microbial metabolites seem to be characteristic of certain biotopes, both on an environmental as well as organismal level. Accordingly, it appears that the search for novel secondary metabolites should center on organisms that inhabit unique biotopes. Thus, it behooves the investigator to carefully study and select the biological source before proceeding, rather than to have a totally random approach in the biological source material. Careful study also indicates that organisms and their biotopes that are subjected to constant metabolic and environmental interactions should produce even more secondary metabolites. Endophytes are microbes that inhabit such biotopes, namely higher plants, which is why they are currently considered as a wellspring of novel secondary metabolites offering the potential for medical, agricultural and/or industrial exploitation. Currently, endophytes are viewed as an outstanding source of bioactive natural products because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. Thus, it would appear that these biotypical factors can be important in plant selection since they may govern the novelty and biological activity of the products associated with endophytic microbes.
Since the discovery of endophytes in Darnel in 1904, various investigators have defined endophytes in different ways which is usually dependent on the perspective from which the endophytes were being isolated and subsequently examined. Bacon and White give an inclusive and widely accepted definition of endophytes—“ Microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects”. While the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests they can also be aggressive saprophytes or opportunistic pathogens. Both fungi and bacteria are the most common microbes existing as endophytes. It would seem that other microbial forms most certainly exist in plants as endophytes, but no evidence for them has yet been presented e.g. mycoplasmas, and archebacteria. The most frequently isolated endophytes are the fungi. It turns out that the vast majority of plants have not been studied for their endophytes. Thus, enormous opportunities exist for the recovery of novel fungal forms, taxa, and biotypes. Hawksworth and Rossman estimated there may be as many as 1 million different fungal species, yet only about 100,000 have been described. As more evidence accumulates, estimates keep rising as to the actual number of fungal species. For instance, Dreyfuss and Chappela estimate there may be at least 1 million species of endophytic fungi alone. It seems obvious that endophytes are a rich and reliable source of genetic diversity and novel, undescribed species. Finally, in our experience, novel microbes usually have associated with them, novel natural products. This fact alone helps eliminate the problems of dereplication in compound discovery.
Rationale for Plant Selection
It is important to understand the methods and rationale used to provide the best opportunities to isolate novel endophytic microorganisms as well as ones making novel bioactive products. Thus, since the number of plant species in the world is so great, creative and imaginative strategies must be used to quickly narrow the search for endophytes displaying bioactivity.
A specific rationale for the collection of each plant for endophyte isolation and natural product discovery is used. Several reasonable hypotheses govern this plant selection strategy and these are as follows:
1. Plants from unique environmental settings, especially those with an unusual biology, and possessing novel strategies for survival are seriously considered for study.
2. Plants that have an ethnobotanical history (use by indigenous peoples) that are related to the specific uses or applications of interest are selected for study. These plants are chosen either by direct contact with local peoples or via local literature. Ultimately, it may be learned that the healing powers of the botanical source, in fact, may have nothing to do with the natural products of the plant, but of the endophyte (inhabiting the plant).
3. Plants that are endemic, having an unusual longevity, or that have occupied a certain ancient land mass, such as Gondwanaland, are also more likely to lodge endophytes with active natural products than other plants.
4. Plants growing in areas of great biodiversity also have the prospect of hosting endophytes with great biodiversity.
Just as plants from a distinct environmental setting are considered to be a promising source of novel endophytes and their compounds, so too are plants with an unconventional biology. For example, an aquatic plant, Rhyncholacis penicillata, was collected from a river system in Southwest Venezuela where the harsh aquatic environment subjected the plant to constant beating by virtue of rushing waters, debris, and tumbling rocks and pebbles. This created many portals through which common phytopathogenic oomycetes could enter the plant. Still, the plant population appeared to be healthy, possibly due to protection from an endophytic product. This was the environmental biological clue used to pick this plant for a comprehensive study of its endophytes. Eventually, a potent antifungal strain of Serratia marcescens, was recovered from R. penicillata and was shown to produce oocydin A, a novel antioomycetous compound having the properties of a chlorinated macrocyclic lactone . It is conceivable that the production of oocydin A by S. marcescens is directly related to the endophyte’s relationship with its higher plant host. Currently, oocydin A is being considered for agriculture use to control the ever threatening presence of oomyceteous fungi such as pythium and phytophthora.
Plants with ethnobotanical history, as mentioned above, also are likely candidates for study since the medical uses to which the plant may have been selected relates more to its population of endophytes than to the plant biochemistry itself. For example, a sample of the snakevine, Kennedia nigriscans, from the Northern Territory of Australia was selected for study since its sap has traditionally been used as bush medicine for many years. In fact, this area was selected for plant sampling since it has been home to the world’s long standing civilization- the Australian Aborigines. The snakevine is harvested, crushed and heated in an aqueous brew by local Aborigines in southwest Arnhemland to treat cuts, wounds and infections. As it turned out, the plant contained a novel endophyte, Streptomyces NRRL 30562, that produces wide spectrum novel peptide antibiotics called- munumbicins. It is reasonable to assume that the healing processes, as discovered by indigenous peoples, might be facilitated by compounds produced by one or more specific plant-associated endophytes as well as the plant products themselves.
In addition, it is worthy to note that some plants generating bioactive natural products have associated endophytes that produce the same natural products. Such is the case with taxol, a highly functionalized diterpenoid and famed anticancer agent that is found in each of the world’s yew tree species (Taxus spp.). In 1993, a novel taxol producing fungus, Taxomyces andreanae, from the yew, Taxus brevifolia was isolated and characterized.
Endophytes and Biodiversity
Of the myriad of ecosystems on earth, those having the greatest biodiveristiy seem to be the ones also having endophytes with the greatest number and the most biodiverse microorganisms. Tropical and temperate rainforests are the most biologically diverse terrestrial ecosystems on earth. The most threatened of these spots cover only 1.44% of the land’s surface, yet, they harbor over 60% of the world’s terrestrial biodiversity. As such, one would expect that areas having high plant endemism also possess specific endophytes that may have evolved with the endemic plant species.
Ultimately, biological diversity implies chemical diversity because of the constant chemical innovation that is exists in ecosystems where the evolutionary race to survive is the most active. Tropical rainforests are a remarkable example of this type of environment. Competition is great, resources are limited and selection pressure is at its peak. This gives rise to a high probability that rainforests are a source of novel molecular structures and biologically active compounds. Bills et al. describe a metabolic distinction between tropical and temperate endophytes through statistical data which compares the number of bioactive natural products isolated from endophytes of tropical regions to the number of those isolated from endophytes of temperate origin. Not only did they find that tropical endophytes provide more active natural products than temperate endophytes, but they also noted that a significantly higher number of tropical endophytes produced a larger number of active secondary metabolites than did fungi from other tropical substrata. This observation suggests the importance of the host plant in influencing the general metabolism of endophytic microbes.
The essence of Jewels of the Jungle is the systematic search of novel microorganisms and their potentially useful products. The film shows how Strobel and his group proceed with the search and what new chemical leads have resulted from these endeavors. The new antimalarial drug-coronamycin was isolated from a small Amazonian vine –known as Monstera sp. The location of this vine and how the novel streptomycete was isolated form this vine is discussed in the film.
Some specific literature references:
1. Strobel, G.A., R.V. Miller, C. Miller, M. Condron, D.B. Teplow, and W.M. Hess. 1999. Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina. Microbiol. 145: 1919-1926.
2. Strobel, G.A. 2002. Rainforest endophytes and bioactive products. Critical Reviews in Biotechnol. 22: 315-333.
3. Daisy, B.H., Strobel, G.A., Castillo, U., Ezra, D., Sears, J., Weaver, D., and Runyon. J.B. 2002. Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel endophytic fungus. Microbiology 148: 3737-3741.
4. Harper, J.K., Ford, E.J. G.A. Strobel, G.A., Arif, A., Grant, D.M., Porco, J., Tomer, D.P. and Oneill, K. 2003. Pestacin: a 1,3 –dihydro isobenzofuran from Pestalotiopsis microspora possessing antioxidant and antimycotic activities. Tetrahedron 59: 2471-2476.
5. Li, J.Y., Sidhu, R.S., Ford, E., Hess, W.M., and Strobel, G.A. 1998. The induction of taxol production in the endophytic fungus - Periconia sp. from Torreya grandifolia. J. Ind. Microbiol. 20: 259-264.
6. Strobel, G.A., Li, J.Y., Sugawara, F., Koshino, H. Harper, J. and Hess, W.M. 1999. Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcescens. Microbiol. 145: 3557-3564.
7. Li, J.Y., Harper, J.K., Grant, D.M., Tombe, B.O., Bashyal, B., Hess, W.M. and Strobel. G.A. 2001. Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. Phytochem. 56: 463-468.
8. Strobel, G.A., Dirksie, E., Sears, J., and Markworth, C. 2001. Volatile antimicrobials from a novel endophytic fungus. Microbiol. 147: 2943-2950.
9. Li, J.Y., Strobel, G.A., Harper, J.K., Lobkovsky, E. and Clardy. J. 2000. Cryptocin, a potent tetramic acid antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina Org. Lett. 2: 767-770.
10. Harrison, L., Teplow, D., M. Rinaldi, M., and Strobel. G.A. 1991. Pseudomycins, a family of novel peptides from Pseudomonas syringae, possessing broad spectrum antifungal activity. J. Gen. Microbiol. 137:2857-2865.
11. Bills, G., Dombrowski, A., Pelaez, F., Polishook, J. and An. Z. 2002. Recent and future discoveries of pharmacologically active metabolites from tropical fungi. p. 165-194. In R. Watling, J.C. Frankland, A.M. Ainsworth, S. Issac, and C.H. Robinson. (ed.), Tropical mycology: micromycetes. vol. 2. CABI Publishing. NY.
12. Ezra, D., Castillo, U., Strobel, G.,. Hess, W.M., Porter, H., Jensen, J., Condron, M., Teplow, D.B., Sears, J, Maranta, M., Hunter, M., Weber, B., and Yaver, D. 2004. Coronamycins, peptide antibiotics produced by a verticillated Streptomyces sp. (MSU-2110) endophytic on Monstera sp. Microbiology. 150: 785-793.
13. NIH. 2001. NIAID global health research plan for HIV/AIDS, malaria and tuberculosis. US Department of Health and Human Services. Bethesda, Md.
www.jewelsofthejungleweb.com/Endophytes.html
Research Scientist - Dr. Gary Strobel Montana State University
Gary A. Strobel was born and raised in Massillon, Ohio. His early years were spent in the Boy Scouting program in his community. In his mid- teens he was awarded both the Eagle badge as well as the W.T. Hornaday Gold medal for his services and promotion of conservation efforts in his community. He completed a B.S. degree at Colorado State University in 1960, and a PhD at the University of California, Davis in 1963. Since that time he has been on the faculty of Montana State University. His research and academic interests have centered on microbe –higher plant relationships. He was co-contributor to the discovery that somaclonal variation occurs in plants and can be used for plant improvement. The discovery of the Ri plasmid in Agrobacterium rhizogenes also originated in his lab. His work on the modification of tree micro flora to preclude plant disease received major national attention in his efforts to biologically control Dutch elm disease. More recently, he has begun to examine endophytic fungi and bacteria for their novel bioactive compounds and their unique biology. He has lectured at over 350 institutes and universities-worldwide and published over 350 articles in scientific journals and holds nearly 50 USA and International patents. He has received numerous awards including an NIH Career Development award, the Wiley award, special recognition from the Royal Nepal Chemical Society and the MSU –VP award for Technology and Science. Recently, he was elected to membership in the American Academy of Microbiology and elected to full fellow of the Explorer’s Club of the World. From 1979-2000 he was chief of the Montana NSF EPSCOR program which encourages and promotes science at all levels of society.
ENDOPHYTES AND THEIR PROMISE FOR NEW MEDICINES AND PRODUCTS FOR AGRICULTURE AND INDUSTRY
Portions of the text taken from: Microbiology and Molecular Biology Reviews 67: 491-502. (2003). This information provides guidelines and rationale for the content of the film---“Jewels of the Jungle”
There is a general call for new antibiotics, chemotherapeutic agents and agrochemicals that are highly effective, possess low toxicity, and will have a minor environmental impact. This search is driven by the development of resistance in infectious microorganisms (e.g. Staphylococcus, Mycobacterium, Streptococcus) to existing compounds and by the menacing presence of naturally resistant organisms. The ingress to the human population of new diseases such as AIDS and SARS requires the discovery and development of new drugs to combat them. Not only do diseases such as AIDS require drugs that target them specifically, but so do new therapies for treating ancillary infections which are a consequence of a weakened immune system. Furthermore, others who are immunocompromised (e.g. cancer and organ transplant patients) are at risk by opportunistic pathogens, such as Aspergillus spp., Cryptococcus spp. and Candida spp., that normally are not major problems in the human population. In addition, more drugs are needed to efficiently treat parasitic protozoan and nematodal infections such as malaria, leshmaniasis, trypanomiasis and filairasis. Malaria alone is more effective in claiming lives each year than any other single infectious agent with the exception of the AIDS virus and Mycobacterium tuberculosis. Finally, because of safety and environmental problems, many synthetic agricultural agents have been, and currently are being targeted for removal from the market, which creates a need to find alternative ways to control farm pests and pathogens. Novel natural products and the organisms that make them offer opportunities for innovation in drug and agrochemical discovery. Exciting possibilities exist for those who are willing to venture into the wild and unexplored territories of the world to experience the excitement and thrill of engaging in the discovery of endophytes, their biology and potential usefulness.
NATURAL PRODUCTS AND TRADITIONAL APPROACHES IN MEDICINE
Natural products are naturally derived metabolites and/or byproducts from microorganisms, plants or animals. These products have been exploited for human use for thousands of years and plants have been the chief source of compounds used for medicine. Even today the largest users of traditional medicines are the Chinese with over 5000 plants, and plant products in their pharmacopoeia. In fact, the world’s best known and most universally used medicinal is aspirin (salicylic acid) which has it natural origins from the glycoside salicin which is found in many species of the plant genera Salix and Populus. Examples abound of natural product use, especially in small native populations in a myriad of remote locations on earth. For instance, certain tribal groups in the Amazon basin, the highland peoples of Papua New Guinea, and the aborigines of Australia each has identified certain plants to provide relief of symptoms varying from head colds to massive wounds and intestinal ailments. History also shows that now extinct civilizations had also discovered the benefits of medicinal plants. In fact, nearly 3000 years ago, the Mayans used fungi grown on roasted green corn to treat intestinal ailments. More recently, the Benedictine monks (800 AD) began to apply Papever somniferum as an anesthetic and pain reliever as the Greeks had done for years before. Many people, in past times, realized that leaf, root and stem concoctions had the potential to help them. These plant products, in general, enhanced the quality of life, reduced pain and suffering, and provided relief, even though an understanding of the chemical nature of bioactive compounds in these complex mixtures and how they functioned remained a mystery.
It was not until Pasteur discovered that fermentation is caused by living cells that people seriously began to investigate microbes as a source for bioactive natural products. Then, scientific serendipity and the power of observation provided the impetus to Fleming to usher in the antibiotic era via the discovery of penicillin from the fungus – Penicillium notatum. Since then, people have been engaged in the discovery and application of microbial metabolites with activity against both plant and human pathogens. Furthermore, the discovery of a plethora of microbes for applications that span a broad spectrum of utility in medicine (e.g. anticancer and immunosuppressant functions), agriculture and industry is now practical because of the development of novel, and sophisticated screening processes in both medicine and agriculture. These processes use individual organisms, cells, enzymes, and site directed techniques, many times in automated arrays, resulting in the rapid detection of promising leads for product development.
Even with untold centuries of human experience behind us and a movement into a modern era of chemistry and automation, it is still the case that natural product based compounds have had an immense impact on modern medicine since about 40% of prescription drugs are based on them. Furthermore, 49% of the new chemical products registered by the FDA are natural products or derivatives thereof. Excluding biologics, between 1989 and1995, 60% of approved drugs and pre-new drug application candidates were of natural origin. From 1983-1994, over 60% of all approved and pre-NDA stage cancer drugs were of natural origin as were 78% of all newly approved antibacterial agents. In fact, the world’s first billion dollar anticancer drug taxol is a natural product derived from the yew tree. Many other examples abound that illustrate the value and importance of natural products in modern civilizations.
Recently, however, natural product research efforts have lost popularity in many major drug companies and, in some cases, have been replaced entirely by combinatorial chemistry which is the automated synthesis of structurally related small molecules. In addition, many drug companies have developed interests in making products that have a larger potential profit base than antiinfectious drugs. These include compounds that provide social benefits, reduce the symptoms of allergies and arthritis, or ones that can sooth the stomach. It appears that this loss of interest can be attributed to the enormous effort and expense that is required to pick, and chose a biological source, then to isolate active natural products, decipher their structures, and begin the long road to product development. It is also apparent that combinatorial chemistry and other synthetic chemistries revolving around certain basic chemical structures is now serving as a never ending source of products to feed the screening robots of the drug industry. Within many large pharmaceutical companies, progress of professionals is primarily based upon numbers of compounds that can be produced and sent to the screening machines. This tends to work against the numerous steps needed even to find one compound in natural product discovery. It seems important to realize that the primary purpose of combinatorial chemistry should be to complement and assist the efforts of natural product drug discovery and development, not to supersede it. The natural product often serves as a lead molecule whose activity can be enhanced by manipulation through combinatorial and synthetic chemistry. Natural products have been the traditional pathfinder compounds with an untold diversity of chemical structures unparalleled by even the largest combinatorial databases.
ENDOPHYTES
It may also be true that a reduction in interest in natural products for use in drug development has happened as a result of people growing weary of dealing with the traditional sources of bioactive compounds including plants of the temperate zones, and microbes from a plethora of soil samples gathered indifferent parts of the world by armies of collectors. In other words, why do something different (working on endophytic microbes) when robots, combinatorial chemistry and molecular biology have arrived on the scene? Furthermore, the logic and rationale for time and effort spent on drug discovery using a target -site directed approach has been overwhelming.
While combinatorial synthesis produces compounds at random, secondary metabolites, defined as low molecular weight compounds not required for growth in pure culture, are produced as an adaptation for specific functions in nature. Shutz notes that certain microbial metabolites seem to be characteristic of certain biotopes, both on an environmental as well as organismal level. Accordingly, it appears that the search for novel secondary metabolites should center on organisms that inhabit unique biotopes. Thus, it behooves the investigator to carefully study and select the biological source before proceeding, rather than to have a totally random approach in the biological source material. Careful study also indicates that organisms and their biotopes that are subjected to constant metabolic and environmental interactions should produce even more secondary metabolites. Endophytes are microbes that inhabit such biotopes, namely higher plants, which is why they are currently considered as a wellspring of novel secondary metabolites offering the potential for medical, agricultural and/or industrial exploitation. Currently, endophytes are viewed as an outstanding source of bioactive natural products because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. Thus, it would appear that these biotypical factors can be important in plant selection since they may govern the novelty and biological activity of the products associated with endophytic microbes.
Since the discovery of endophytes in Darnel in 1904, various investigators have defined endophytes in different ways which is usually dependent on the perspective from which the endophytes were being isolated and subsequently examined. Bacon and White give an inclusive and widely accepted definition of endophytes—“ Microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects”. While the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests they can also be aggressive saprophytes or opportunistic pathogens. Both fungi and bacteria are the most common microbes existing as endophytes. It would seem that other microbial forms most certainly exist in plants as endophytes, but no evidence for them has yet been presented e.g. mycoplasmas, and archebacteria. The most frequently isolated endophytes are the fungi. It turns out that the vast majority of plants have not been studied for their endophytes. Thus, enormous opportunities exist for the recovery of novel fungal forms, taxa, and biotypes. Hawksworth and Rossman estimated there may be as many as 1 million different fungal species, yet only about 100,000 have been described. As more evidence accumulates, estimates keep rising as to the actual number of fungal species. For instance, Dreyfuss and Chappela estimate there may be at least 1 million species of endophytic fungi alone. It seems obvious that endophytes are a rich and reliable source of genetic diversity and novel, undescribed species. Finally, in our experience, novel microbes usually have associated with them, novel natural products. This fact alone helps eliminate the problems of dereplication in compound discovery.
Rationale for Plant Selection
It is important to understand the methods and rationale used to provide the best opportunities to isolate novel endophytic microorganisms as well as ones making novel bioactive products. Thus, since the number of plant species in the world is so great, creative and imaginative strategies must be used to quickly narrow the search for endophytes displaying bioactivity.
A specific rationale for the collection of each plant for endophyte isolation and natural product discovery is used. Several reasonable hypotheses govern this plant selection strategy and these are as follows:
1. Plants from unique environmental settings, especially those with an unusual biology, and possessing novel strategies for survival are seriously considered for study.
2. Plants that have an ethnobotanical history (use by indigenous peoples) that are related to the specific uses or applications of interest are selected for study. These plants are chosen either by direct contact with local peoples or via local literature. Ultimately, it may be learned that the healing powers of the botanical source, in fact, may have nothing to do with the natural products of the plant, but of the endophyte (inhabiting the plant).
3. Plants that are endemic, having an unusual longevity, or that have occupied a certain ancient land mass, such as Gondwanaland, are also more likely to lodge endophytes with active natural products than other plants.
4. Plants growing in areas of great biodiversity also have the prospect of hosting endophytes with great biodiversity.
Just as plants from a distinct environmental setting are considered to be a promising source of novel endophytes and their compounds, so too are plants with an unconventional biology. For example, an aquatic plant, Rhyncholacis penicillata, was collected from a river system in Southwest Venezuela where the harsh aquatic environment subjected the plant to constant beating by virtue of rushing waters, debris, and tumbling rocks and pebbles. This created many portals through which common phytopathogenic oomycetes could enter the plant. Still, the plant population appeared to be healthy, possibly due to protection from an endophytic product. This was the environmental biological clue used to pick this plant for a comprehensive study of its endophytes. Eventually, a potent antifungal strain of Serratia marcescens, was recovered from R. penicillata and was shown to produce oocydin A, a novel antioomycetous compound having the properties of a chlorinated macrocyclic lactone . It is conceivable that the production of oocydin A by S. marcescens is directly related to the endophyte’s relationship with its higher plant host. Currently, oocydin A is being considered for agriculture use to control the ever threatening presence of oomyceteous fungi such as pythium and phytophthora.
Plants with ethnobotanical history, as mentioned above, also are likely candidates for study since the medical uses to which the plant may have been selected relates more to its population of endophytes than to the plant biochemistry itself. For example, a sample of the snakevine, Kennedia nigriscans, from the Northern Territory of Australia was selected for study since its sap has traditionally been used as bush medicine for many years. In fact, this area was selected for plant sampling since it has been home to the world’s long standing civilization- the Australian Aborigines. The snakevine is harvested, crushed and heated in an aqueous brew by local Aborigines in southwest Arnhemland to treat cuts, wounds and infections. As it turned out, the plant contained a novel endophyte, Streptomyces NRRL 30562, that produces wide spectrum novel peptide antibiotics called- munumbicins. It is reasonable to assume that the healing processes, as discovered by indigenous peoples, might be facilitated by compounds produced by one or more specific plant-associated endophytes as well as the plant products themselves.
In addition, it is worthy to note that some plants generating bioactive natural products have associated endophytes that produce the same natural products. Such is the case with taxol, a highly functionalized diterpenoid and famed anticancer agent that is found in each of the world’s yew tree species (Taxus spp.). In 1993, a novel taxol producing fungus, Taxomyces andreanae, from the yew, Taxus brevifolia was isolated and characterized.
Endophytes and Biodiversity
Of the myriad of ecosystems on earth, those having the greatest biodiveristiy seem to be the ones also having endophytes with the greatest number and the most biodiverse microorganisms. Tropical and temperate rainforests are the most biologically diverse terrestrial ecosystems on earth. The most threatened of these spots cover only 1.44% of the land’s surface, yet, they harbor over 60% of the world’s terrestrial biodiversity. As such, one would expect that areas having high plant endemism also possess specific endophytes that may have evolved with the endemic plant species.
Ultimately, biological diversity implies chemical diversity because of the constant chemical innovation that is exists in ecosystems where the evolutionary race to survive is the most active. Tropical rainforests are a remarkable example of this type of environment. Competition is great, resources are limited and selection pressure is at its peak. This gives rise to a high probability that rainforests are a source of novel molecular structures and biologically active compounds. Bills et al. describe a metabolic distinction between tropical and temperate endophytes through statistical data which compares the number of bioactive natural products isolated from endophytes of tropical regions to the number of those isolated from endophytes of temperate origin. Not only did they find that tropical endophytes provide more active natural products than temperate endophytes, but they also noted that a significantly higher number of tropical endophytes produced a larger number of active secondary metabolites than did fungi from other tropical substrata. This observation suggests the importance of the host plant in influencing the general metabolism of endophytic microbes.
The essence of Jewels of the Jungle is the systematic search of novel microorganisms and their potentially useful products. The film shows how Strobel and his group proceed with the search and what new chemical leads have resulted from these endeavors. The new antimalarial drug-coronamycin was isolated from a small Amazonian vine –known as Monstera sp. The location of this vine and how the novel streptomycete was isolated form this vine is discussed in the film.
Some specific literature references:
1. Strobel, G.A., R.V. Miller, C. Miller, M. Condron, D.B. Teplow, and W.M. Hess. 1999. Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina. Microbiol. 145: 1919-1926.
2. Strobel, G.A. 2002. Rainforest endophytes and bioactive products. Critical Reviews in Biotechnol. 22: 315-333.
3. Daisy, B.H., Strobel, G.A., Castillo, U., Ezra, D., Sears, J., Weaver, D., and Runyon. J.B. 2002. Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel endophytic fungus. Microbiology 148: 3737-3741.
4. Harper, J.K., Ford, E.J. G.A. Strobel, G.A., Arif, A., Grant, D.M., Porco, J., Tomer, D.P. and Oneill, K. 2003. Pestacin: a 1,3 –dihydro isobenzofuran from Pestalotiopsis microspora possessing antioxidant and antimycotic activities. Tetrahedron 59: 2471-2476.
5. Li, J.Y., Sidhu, R.S., Ford, E., Hess, W.M., and Strobel, G.A. 1998. The induction of taxol production in the endophytic fungus - Periconia sp. from Torreya grandifolia. J. Ind. Microbiol. 20: 259-264.
6. Strobel, G.A., Li, J.Y., Sugawara, F., Koshino, H. Harper, J. and Hess, W.M. 1999. Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcescens. Microbiol. 145: 3557-3564.
7. Li, J.Y., Harper, J.K., Grant, D.M., Tombe, B.O., Bashyal, B., Hess, W.M. and Strobel. G.A. 2001. Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. Phytochem. 56: 463-468.
8. Strobel, G.A., Dirksie, E., Sears, J., and Markworth, C. 2001. Volatile antimicrobials from a novel endophytic fungus. Microbiol. 147: 2943-2950.
9. Li, J.Y., Strobel, G.A., Harper, J.K., Lobkovsky, E. and Clardy. J. 2000. Cryptocin, a potent tetramic acid antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina Org. Lett. 2: 767-770.
10. Harrison, L., Teplow, D., M. Rinaldi, M., and Strobel. G.A. 1991. Pseudomycins, a family of novel peptides from Pseudomonas syringae, possessing broad spectrum antifungal activity. J. Gen. Microbiol. 137:2857-2865.
11. Bills, G., Dombrowski, A., Pelaez, F., Polishook, J. and An. Z. 2002. Recent and future discoveries of pharmacologically active metabolites from tropical fungi. p. 165-194. In R. Watling, J.C. Frankland, A.M. Ainsworth, S. Issac, and C.H. Robinson. (ed.), Tropical mycology: micromycetes. vol. 2. CABI Publishing. NY.
12. Ezra, D., Castillo, U., Strobel, G.,. Hess, W.M., Porter, H., Jensen, J., Condron, M., Teplow, D.B., Sears, J, Maranta, M., Hunter, M., Weber, B., and Yaver, D. 2004. Coronamycins, peptide antibiotics produced by a verticillated Streptomyces sp. (MSU-2110) endophytic on Monstera sp. Microbiology. 150: 785-793.
13. NIH. 2001. NIAID global health research plan for HIV/AIDS, malaria and tuberculosis. US Department of Health and Human Services. Bethesda, Md.
www.jewelsofthejungleweb.com/Endophytes.html
Research Scientist - Dr. Gary Strobel Montana State University
Gary A. Strobel was born and raised in Massillon, Ohio. His early years were spent in the Boy Scouting program in his community. In his mid- teens he was awarded both the Eagle badge as well as the W.T. Hornaday Gold medal for his services and promotion of conservation efforts in his community. He completed a B.S. degree at Colorado State University in 1960, and a PhD at the University of California, Davis in 1963. Since that time he has been on the faculty of Montana State University. His research and academic interests have centered on microbe –higher plant relationships. He was co-contributor to the discovery that somaclonal variation occurs in plants and can be used for plant improvement. The discovery of the Ri plasmid in Agrobacterium rhizogenes also originated in his lab. His work on the modification of tree micro flora to preclude plant disease received major national attention in his efforts to biologically control Dutch elm disease. More recently, he has begun to examine endophytic fungi and bacteria for their novel bioactive compounds and their unique biology. He has lectured at over 350 institutes and universities-worldwide and published over 350 articles in scientific journals and holds nearly 50 USA and International patents. He has received numerous awards including an NIH Career Development award, the Wiley award, special recognition from the Royal Nepal Chemical Society and the MSU –VP award for Technology and Science. Recently, he was elected to membership in the American Academy of Microbiology and elected to full fellow of the Explorer’s Club of the World. From 1979-2000 he was chief of the Montana NSF EPSCOR program which encourages and promotes science at all levels of society.