Final Report Summary - GAMEXP (Genomic approaches to metabolite exploitation from Xenorhabdus, Photorhabdus)
Executive summary:
GAMEXP has shown the great potential of Xenorhabdus / Photorhabdus bacteria as novel producers of bioactive natural products and thus has put these unexplored genera at the same level as well-known natural product producers like Streptomyces, Bacillus or Myxobacteria. The goal in the consortium of six partners from Thailand, Vietnam, the United Kingdom (UK), and Germany, was to have as many natural products as possible specifically and probably unique from these bacteria in order to test them initially within GAMEXP but also to allow their testing in more detail and also against additional targets by pharmaceutical companies after the duration of GAMEXP.
The main activities and achievements within GAMEXP were:
- isolation of bacteria from soil samples collected in Vietnam and Thailand
- 1448 soil samples have been collected and from these
- more than 200 Xenorhabdus / Photorhabdus strains have been isolated
- this is probably one of the largest collection of Xenorhabdus / Photorhabdus strains in the world
- all isolated strains and several other strains obtained from lab and public strain collections were analysed chemically
- more than 500 compounds have been identified in these strains using mass spectrometry, most of them unique to Xenorhabdus / Photorhabdus
- a database has been built for Xenorhabdus / Photorhabdus specific compounds
- 187 compounds have been isolated or have been synthesised after their structure was fully solved by mass spectrometry
- several new compound classes and new derivatives of known compound classes have been identified
- the pure compounds were tested against several different target organisms including bacteria, fungi, protozoa, insects
- several bioactive compounds have been identified with antibiotic activity
- several active compounds against neglected tropical diseases have been identified
- more than 15 genomes of Xenorhabdus / Photorhabdus strains have been analysed for biosynthesis gene clusters involved in natural product biosynthesis
- more than 20 of these gene clusters have been analysed in detail
- molecular tools for accessing this chemical diversity have been established in order to increase the production of these compounds
- the scientific community has been pointed to GAMEXP
- more than 24 scientific publications in peer reviewed journals have been written
- a patent has been filed about the most abundant and novel compound class from Xenorhabdus / Photorhabdus
- more than 70 additional dissemination activities (talks, posters, newspaper articles) have been done
- pharmaceutical and chemical companies have been attracted to the results obtains during GAMEXP
Project context and objectives:
The proposed project (GAMEXP) combined European expertise in natural product chemistry, microbiology and molecular biology to isolate new small molecules for medicine.
75 % of all compounds used to treat infectious diseases are either natural products, derived from natural products or are inspired from natural products (NP). This is even more astonishing since many more synthetic organic compounds are known than compounds derived from natural sources. However, it has been shown that natural products cover a different chemical space compared to synthetic compounds and often break the rule of five that refers to the chemical properties of medically used synthetic compounds that are indicative of their drugability. The major sources for natural compounds currently in use are actinomycetes bacteria which have been investigated throughout within the last 60 years. Although new compounds with interesting biological activities can still be isolated, the rate of re-isolating already known compounds is high. To solve this problem, new sources for bioactive compounds have to be identified. This is of utmost importance since we are confronted with a plethora of emerging and re-emerging diseases all over the world (e.g. tuberculosis, plague, melioidosis, human ehrlichioses, tularaemia and trench fever) and because of the increasing resistance of several pathogenic bacteria (e.g. multi-resistant Staphylococcus aureus MRSA and Mycobacterium strains) even against the last border antibiotics (e.g. vancomycin-resistant enterococci). Here natural products seem to be a valuable resource as they have been developed in nature within a biological context that might already imply a high proportion of compounds with a biological activity against different targets. Although it is still not clear what the function of all NPs in the natural ecosystem is six, it is clear for some of them that they function as chemical weapons that should kill and/or suppress competitors. With respect to this function it is not surprising that several weapon-like antibiotics have been identified from bacteria living in highly competitive environments like soil.
The great dilemma of anti-infective research is that almost all big pharmaceutical companies have stopped their research and development of new compounds as there is a profit problem in antibiotic R&D. Currently the interest in NPs seems to increase again which is also due to special programmes that support such research by pharmaceutical companies and because of our desperate need for these compounds. Without new anti-infectives we are rapidly approaching a situation prior to World War II where millions of people died of infectious diseases that are still curable at the moment but might not be in 5 - 10 years. As a result of this frightening development, pharmaceutical companies and biotech companies in the United States (US) are encouraged by tax cuts and long lasting patents when they develop new anti-infectives.
Expert knowledge in nematode sampling and characterisation of their symbiotic bacteria from Thailand and Vietnam was used to address the topic of bioprospecting Xenorhabdus and Photorhabdus. These drug producing bacteria live in symbiosis with entomopathogenic nematodes and are used by the nematodes to kill their insect hosts. Although these bacteria are proven producers of natural products, only a few compounds have been isolated and studied in-depth. The isolated natural products were used to build up a unique Xenorhabdus / Photorhabdus-specific compound library which was tested as drug leads for a variety of infectious diseases including anti-protozoal, anti-malaria, anthelminthic, and antibiotic activity. Additionally, insects were used as infection models in order to reduce the need for mammalian testing in preclinical analysis. The complete bioactivity data set for any given compound was collected as basis for future exploitation with pharmaceutical companies. Moreover, the most promising Xenorhabdus / Photorhabdus strains were used in an extensive molecular biology program that would finally result in E. coli strains expressing the biosynthesis pathways responsible for the bioactivity of interest or in Xenorhabdus / Photorhabdus strains over-producing the desired compounds. Finally, the compound library with its connected bioactivity data was made accessible to pharmaceutical companies.
The ultimate goals of the project were therefore the following:
- The isolation of a diverse and large collection of Xenorhabdus / Photorhabdus strains from entomopathogenic nematodes.
- The isolation of new compounds from Xenorhabdus / Photorhabdus strains in order to build up a Xenorhabdus / Photorhabdus-specific compound library that would prove the great potential of Xenorhabdus / Photorhabdus for secondary metabolites.
- Multiple and parallel bioactivity testing against bacteria, fungi, protozoa, nematodes, insects and mammalian cytotoxicity of the compound library.
- Genome mining of the five most promising strains including construction of cosmid / BAC libraries, RVA screening, genome sequencing and heterologous expression in E. Coli.
- In order to attract industrial partners after the duration of the project, all legal issues such as IPR, international agreements and access rights will be settled in advance of licensing the products to companies.
In order to achieve these goals, the project was subdivided into seven work packages (WP1 - WP7). WP1 - WP4 were the core WPs.
WP1 (Strain isolation/characterisation) was the basis for the whole project. A large and diverse strain collection of Xenorhabdus / Photorhabdus strains was obtained from different locations. The work in WP1 was carried out mostly by experts in nematology and microbiology from Thailand and Vietnam.
In WP2 (Chemistry / compound library) all Xenorhabdus / Photorhabdus strains were analysed using analytical methods and new compounds are isolated in a preparative scale from large scale cultivations or synthesized after their structure elucidation to build up a Xenorhabdus / Photorhabdus-specific compound library. This work was carried out by expert natural product chemists in Germany.
These compounds were analyzed in WP3 (Bioactivity testing) against multiple different targets (bacteria, fungi, protozoa, nematodes, insects, cell cultures) in parallel. In order to rapidly identify interesting strains, crude extracts were also tested using the same assays. This work was carried out by biologists well familiar with these standard assays in the UK.
The aim of WP4 (Genome mining) was to identify the biosynthesis gene clusters responsible for the production of the desired compounds allowing their future exploitation and enabling the heterologous production of these compounds in recombinant E. coli. This work was carried out by partners in the UK and Germany.
WP5 (Provision of harmonised set of methodologies) consisted of two sessions. The initial session was required to ensure that all beneficiaries use the same or a comparable methodology regarding especially the bioactivity testing. The consolidation session ensured that the used methods are still comparable but should also involve methods used within WP4 (genome mining).
WP6 (Innovation-related activities) included management of all GAMEXP related intellectual property right issues and coordination of dissemination activities, including the development of the overall dissemination strategy towards the scientific community and the broader public.
WP7 (Management) included the overall responsibility for managing the different work package activities, monitoring timely submission of deliverables and the overall project progress to fulfill the tasks and objectives set out in the work programme, collecting data and preparing interim reports.
Project results:
The following describes selected examples of the results obtains during GAMEXP. As most of the results will be used in final publications that the GAMEXP consortium is currently writing or is sensitive and will be used for additional patents, the presented examples are taken from work already published as part of GAMEXP. A much more detailed description of the GAMEXP results can be found as part of the different deliverables as well as the periodic reports.
In WP1 (Strain isolation and characterisation) more than 1400 soil samples have been collected in Thailand and Vietnam and more than 200 insect-pathogenic Xenorhabdus / Photorhabdus strains have been isolated from these soil samples using an insect baiting technique. All bacterial strains have been analysed using colony morphology as well as recA sequence as morphological marker. Additionally, the bacteria also several of the corresponding entomopathogenic nematode (EPN) partners have been collected and identified using morphological and molecular markers.
In WP2 (Chemistry / compound library) all Xenorhabdus / Photorhabdus strains from Thailand and Vietnam as well as additional strains obtained from public strain collections (DSMZ, ATCC) as well as from other labs working with Xenorhabdus / Photorhabdus, were cultivated on a small scale and the cultures were extracted for subsequent analysis using HPLC coupled to mass spectrometry (HPLC/MS). Here the plan was originally to identify 50 - 100 compounds. However, Xenorhabdus / Photorhabdus were so potent producers of novel chemical diversity, that more than 500 compounds were identified in these strains, most of them unique to Xenorhabdus / Photorhabdus. A MS-MS fragmentation-based database has been set up in order to identify these known compounds but also to identify even more novel compounds that can be analysed in the future.
In order to elucidate the structure of peptides among these compounds based on MS experiments, a new method was developed allowing the determination of the amino acid stereochemistry via a combination of labelling and detailed MS experiments (DOI: 10.1002/chem.201103479) as exemplified for the GAMEXPeptides found in Photorhabdus luminescens. Also as part of the GAMEXP search for novel compounds from Xenorhabdus / Photorhabdus a novel prodrug activation mechanism for non-ribosomal peptides has been identified (DOI: 10.1038/nchembio.688).
A major goal of WP2 was also to build a Xenorhabdus / Photorhabdus specific compound library and at the end this library contains 187 Xenorhabdus / Photorhabdus compounds (these compounds obtained a unique HB# number). This large number of compounds has either been isolated from large scale cultivation of the respective strains or has been synthesized chemically after their structure has been solved by MS and labelling experiments as described above. For peptides, the synthesis is often superior to the isolation of the natural product, especially when the peptides are only produced in very small amounts by the original producer. Among the novel compounds also part of the compound library are the simple phenylethylamides showing an interesting biological activity (DOI: 10.1002/cbic.201100223) just to give one already published example.
The identification of all these compounds based on HPLC/MS also allowed the correlation between phylogeny and compounds present in the individual strains revealing the presence of several highly conserved natural products but also compounds being specific for a subclade of strains or present only in a few species.
Besides HPLC/ESI-MS also MALDI-MS was used for the identification of compounds, which turned out to be especially powerful for very large hydrophobic peptides and highly polar peptides containing several arginine moieties (DOI:10.1021/ac300372p).
In WP3 (Bioactivity testing) the pure compounds were tested against bacteria, fungi, nematodes, protozoa including species causing tropical neglected diseases, insects and insect cells and other eukaryotic cell lines. Here especially antibiotically active compounds have been identified of which some were new or new derivatives of known compound classes (like the xenorhabdins and the xenocoumacins). Even more important, several compounds showed an activity and or selectivity against neglected tropical diseases as tested by the Swiss Tropical and Public Health Institute (Swiss TPH). Among them structurally novel peptide classes but also several low molecular weight compounds. The activity of a few compound classes was in fact so good that further tests are planned in order to evaluate these compounds also in vivo.
When using insect cells as target, microscopic high content analysis was performed as shown for two compounds. Here, differences could be observed even no cytotoxicity could be obtained in some cases.
In WP4 (Genome mining) 15 strains of Xenorhabdus / Photorhabdus were analysed for the presence of biosynthesis gene clusters involved in the production of natural products. Thus, the genomes of several Xenorhabdus / Photorhabdus strains selected based on the production of several different compound classes or the production of conserved and/or bioactive compound classes have been sequenced. Although most genomes were not closed, from the contig data the biosynthesis gene clusters could be identified. Here, we could identify the biosynthesis gene clusters of the most promising bioactive compound class (against tropical neglected diseases) were identified in several different genomes finally resulting in the submission of a patent in order to protect these genes, the corresponding proteins as well as the compounds derived thereof. Additionally, more than 200 other biosynthesis gene clusters have been identified. Several of these have been assigned to a biological activity in the rapid virulence annotation (RVA) approach.
15 biosynthesis gene clusters have been studied in either of the following ways:
- Genes being part of the biosynthesis gene were disrupted or deleted in the original producer, resulting in the production of modified derivatives. This led to the identification of several novel biosynthesis mechanisms, which will also be studied in detail in the future.
- Biosynthesis gene clusters were expressed heterologously in E. coli, resulting in the production of derivatives of the natural products and/or in higher yields.
- The native promoter was exchanged against strong constitutive or inducible promoters in the native producer resulting in the activation of previously silent biosynthesis gene clusters and thus also in the production of the corresponding natural products for the first time (DOI:10.1016/j.jbiotec.2011.10.002).
- The same approach was also very efficient in increasing the yields of already known compounds.
In order to achieve all these goals methods for promoter exchange approach or the hetereologous expression have been developed and/or optimized for Xenorhabdus / Photorhabdus.
For Photorhabdus asymbiotica, which is not only entomopathogenic but can also infect mammals and thus is also an emerging human pathogen, the transcriptome was analysed revealing the presence of several virulence islands activated in human serum or at 37 °C, the temperature of the human body. Here RNA seq methodologies were performed which gave a huge but very robust data set that will also be analyzed in the future. A first result was that also some natural product biosynthesis gene clusters were differentially regulated pointing to the corresponding compounds as virulence factors. Subsequently, compounds were identified, that inhibits the human immune system and thus might play an important part in the pathogenicity of P. asymbiotica.
In order to ensure that all partners use the same methods and know all methods used within GAMEXP, two method manuals (Deliverable D5.1 and D5.2) were written as part of WP5 (Provision of harmonised set of methodologies). D5.1 was dedicated to the isolation of nematodes and bacteria from soil samples and D5.2 was dedicated to all aspects of genome mining used during GAMEXP. Due to the fact that novel tools for genome mining (promoter exchange approach, efficient heterologous expression), D5.2 was updated during GAMEXP.
As part of WP6 (Innovation-related activities) a website for GAMEXP was established (see http://www.GAMEXP.eu(odnośnik otworzy się w nowym oknie) online), allowing also the establishment of the communication structures and its use as data repository for reports and publications. As part of WP6, all intellectual property rights (IPR) issues were solved regarding strains and nematodes or natural products derived thereof. Additionally, SWOT analyses and PUDK analysis were an integral part of WP6.
The management of GAMEXP (WP7) was performed by the company EURICE, specialised in EC-project management. This included the organisation of different project meetings as well as the preparation and submission of project reports.
Potential impact:
-The time has come to close the book of infectious diseases stated by the US General Surgeon William H. Stewart in 1969 has been proven fatally wrong. This statement was given when antibiotic research and discovery had its zenith and new classes of potent drugs entered the market, resulting in the disappearance of many formerly deadly infectious diseases at least in the industrialised nations.
Infectious diseases, however, have made a stunning comeback. Syphilis, gonorrhea, and tuberculosis are all re-emerging with vengeance and the advent of AIDS, hepatitis C, and severe acute respiratory syndrome (SARS) has been a humbling experience. However, the less publicised trend of bacterial resistance may pose an even greater threat. Statistics from WHO hint as to what the future may hold: Vibrio cholerae is 100 % resistant to both tetracycline and chloramphenicol in Tanzania and Rwanda; in some regions, penicillin-resistant Streptococcus pneumoniae makes up 70 % of strains; worldwide, multidrug-resistant Mycobacterium tuberculosis accounts for 1 to 22 % of all new cases. In the US, methicillin-resistant Staphylococcus aureus now accounts for nearly 60 % of hospital-acquired staphylococcal infections, and 20 % of nosocomial infections in US hospitals are reported to be multidrug resistant. Although the numbers are slightly better in Europe, unfortunately Europe is catching up fast.
Even without multidrug resistant strains, nosocomial infections are increasingly important as it has been shown that nearly USD 5 billion are added to US health costs every year as a result of infections that patients get while they are hospitalized for other health problems.
Furthermore, it is reported that that nearly 2 million patients annually acquire infections while being treated for other illness or injury in hospital, and nearly 88 000 die as a direct or indirect result of this infection (4th Decennial Conference on Nosocomial and Health-care associated Infections, Atlanta US). This means, that approximately 1 in 10 hospitalised patients will acquire an infection after admission (Emerging infectious diseases, Vol. 10, No. 4, April 2004). The extra costs arise largely due to the extra days that the patient has to spend in hospital. In general the number of days that a patient has to spend in the hospital varies depending on the type of infection he or she gets: an estimated 1 to 4 days for urinary tract infections, 7 to 8 days for infection at the site of surgery procedure, 7 to 21 days for bloodstream infections and 7 to 30 days for pneumonia. The costs vary from anywhere like USD 600 for urinary tract infections to USD 5000 or more for pneumonia. Prolonged bloodstream infections can top USD 50 000. Other sources reported, that the annual economic costs amount to USD 6,7 billion per year in the US and approximately USD 1,7 billion in the UK indicating that nosocomial infections are in fact in a global problem similarly as infections in general are a global problem.
Even with the adoption of drugs with novel mechanisms of action, we do not have to ask the question if resistance will develop but only when it will occur. This is simply the consequence of the enormous speed at which microorganisms exchange and mutate their genes. It is predictable that the winner in this game will always be the microbe, simply because of its huge numbers, short generation times, and mutation rates.
The best way to combat antimicrobial resistance or at least delay it, is by reducing or, preferably, eliminating the misuse of antibiotics in medicine and agriculture. Moreover, we also have to find new drugs with new mode of actions and therefore new sources of these drugs have to be identified as it is the goal of the current call HEALTH 2.3.4-3 in which this proposal is submitted.
A number of genome sequencing projects within the last five years have revealed several bacterial species that seem to be capable to produce secondary metabolites. Instead the isolation of new species of well-known natural product producers (e.g. actinomycetes) from extreme or not-yet explored environments (e.g. deep sea), one way might be to bioprospect bacterial species that seem to be good NP producers (e.g. Burkholderia, Xenorhabdus, Photorhabdus).
Improving health in general is important in its own right. But is also an important part of the solution to address a number of key challenges facing Europe such as population ageing, security threats or labour shortage. Obviously, infectious diseases play a major role within all these challenges and therefore the question is how bioprospecting projects such as GAMEXP might be involved in their solution?
Possible answers to this can be the following long-term benefits which might arise directly or indirectly from this project:
1. New anti-infective compounds: In an ideal case a compound found within this project is developed into a pharmaceutical product within the next 10 years by a pharmaceutical company to treat one or several diseases.
2. The methods applied within this project (activation of silent biosynthesis gene clusters to increase the chemical diversity) are used for the identification of anti-infective compounds from other organisms.
3. The identification of several biologically active compounds from Xenorhabdus / Photorhabdus will stimulate other research groups or even pharmaceutical companies to explore this huge resource and thereby might lead to the identification of desperately needed cures.
As all current EU problems with infectious diseases are mirrored in countries all over the world, bioprospecting in general will also benefit global health issues, e.g. in Thailand and Vietnam and other developing countries with important tropical diseases (e.g. melioidosis, malaria).
Here it is noteworthy that one of us (Dr Aunchalee Thanwisai who obtained her PhD within GAMEXP) has decided to start her independent career in the field of Xenorhabdus / Photorhabdus in Thailand.
With the aim of expediting the development of new diagnostics, drugs, vaccines and tools for transmission control for effective prevention, treatment and control, the GAMEXP consortium will be established to address several infectious diseases including selected neglected diseases. The proposed work will establish bacteria of the genera Xenorhabdus and Photorhabdus as new source for natural products with potential biological activity. Compounds should be isolated resulting in a library Xenorhabdus / Photorhabdus-specific compounds which are then analyzed in bioactivity tests against a plethora of infectious diseases including several emerging and neglected diseases. This would include assays against multi-resistant Gram-negative and positive bacteria (Vibrio, Pseudomonas, Staphylococcus, Mycobacterium) as well as assay against emerging human bacterial infections (e.g. Burkholderia). Moreover, assays will be conducted against opportunistic pathogenic fungi or yeasts (e.g. Candida involved in AIDS), Acantamoeba as model-protozoa to detect compounds useful to treat trypanosomiasis or leishmaniasis, C. elegans as model for anthelminthic compounds to treat river blindness and filiariasis. Additionally, cytotoxicity against mammalian cell cultures and insects will be determined as model organisms for higher organism.
The compounds with the most promising bioactivity data will be protected. Furthermore, in order to truly establish Xenorhabdus / Photorhabdus as new source of natural products genome mining will be performed that will result also in the finding of the biosynthesis gene clusters responsible for the production of the compounds of interest. This is a prerequisite for biotechnological exploitation of these bacteria.
The unique approach of combining rapid chemical identification, genome mining and massively parallel bioactivity testing in non-animal models will vastly increase the current speed of drug discovery and development. The rigorous patent strategy will also attract partners at an early stage in order to take-over novel bioactives for drug development and deployment.
Regarding general dissemination activities 24 publications in peer-reviewed scientific journals have already been published, three more are already accepted or in press.
Additionally, one patent on the most abundant and novel class of compounds from Xenorhabdus / Photorhabdus showing activity against neglected tropical diseases has been filed. 78 additional dissemination activities (web releases, press releases, newspaper articles) were performed as part of GAMEXP among them 45 talks and presentations given as well as 14 posters presented by GAMEXP members at national and international conferences and meetings.
Project website: http://www.gamexp.eu(odnośnik otworzy się w nowym oknie)
Contact:
Prof. Dr Helge B. Bode,
Goethe Universitat Frankfurt, Germany,
Institut fur Molekulare Biowissenschaften,
Biozentrum/Campus Riedberg (N250, EG),
Max-von-Laue-Str. 9,
60438 Frankfurt am Main
Tel: +49 69 798 29522
Fax: +49 69 798 29527
E-mail: h.bode@bio.uni-frankfurt.de
GAMEXP has shown the great potential of Xenorhabdus / Photorhabdus bacteria as novel producers of bioactive natural products and thus has put these unexplored genera at the same level as well-known natural product producers like Streptomyces, Bacillus or Myxobacteria. The goal in the consortium of six partners from Thailand, Vietnam, the United Kingdom (UK), and Germany, was to have as many natural products as possible specifically and probably unique from these bacteria in order to test them initially within GAMEXP but also to allow their testing in more detail and also against additional targets by pharmaceutical companies after the duration of GAMEXP.
The main activities and achievements within GAMEXP were:
- isolation of bacteria from soil samples collected in Vietnam and Thailand
- 1448 soil samples have been collected and from these
- more than 200 Xenorhabdus / Photorhabdus strains have been isolated
- this is probably one of the largest collection of Xenorhabdus / Photorhabdus strains in the world
- all isolated strains and several other strains obtained from lab and public strain collections were analysed chemically
- more than 500 compounds have been identified in these strains using mass spectrometry, most of them unique to Xenorhabdus / Photorhabdus
- a database has been built for Xenorhabdus / Photorhabdus specific compounds
- 187 compounds have been isolated or have been synthesised after their structure was fully solved by mass spectrometry
- several new compound classes and new derivatives of known compound classes have been identified
- the pure compounds were tested against several different target organisms including bacteria, fungi, protozoa, insects
- several bioactive compounds have been identified with antibiotic activity
- several active compounds against neglected tropical diseases have been identified
- more than 15 genomes of Xenorhabdus / Photorhabdus strains have been analysed for biosynthesis gene clusters involved in natural product biosynthesis
- more than 20 of these gene clusters have been analysed in detail
- molecular tools for accessing this chemical diversity have been established in order to increase the production of these compounds
- the scientific community has been pointed to GAMEXP
- more than 24 scientific publications in peer reviewed journals have been written
- a patent has been filed about the most abundant and novel compound class from Xenorhabdus / Photorhabdus
- more than 70 additional dissemination activities (talks, posters, newspaper articles) have been done
- pharmaceutical and chemical companies have been attracted to the results obtains during GAMEXP
Project context and objectives:
The proposed project (GAMEXP) combined European expertise in natural product chemistry, microbiology and molecular biology to isolate new small molecules for medicine.
75 % of all compounds used to treat infectious diseases are either natural products, derived from natural products or are inspired from natural products (NP). This is even more astonishing since many more synthetic organic compounds are known than compounds derived from natural sources. However, it has been shown that natural products cover a different chemical space compared to synthetic compounds and often break the rule of five that refers to the chemical properties of medically used synthetic compounds that are indicative of their drugability. The major sources for natural compounds currently in use are actinomycetes bacteria which have been investigated throughout within the last 60 years. Although new compounds with interesting biological activities can still be isolated, the rate of re-isolating already known compounds is high. To solve this problem, new sources for bioactive compounds have to be identified. This is of utmost importance since we are confronted with a plethora of emerging and re-emerging diseases all over the world (e.g. tuberculosis, plague, melioidosis, human ehrlichioses, tularaemia and trench fever) and because of the increasing resistance of several pathogenic bacteria (e.g. multi-resistant Staphylococcus aureus MRSA and Mycobacterium strains) even against the last border antibiotics (e.g. vancomycin-resistant enterococci). Here natural products seem to be a valuable resource as they have been developed in nature within a biological context that might already imply a high proportion of compounds with a biological activity against different targets. Although it is still not clear what the function of all NPs in the natural ecosystem is six, it is clear for some of them that they function as chemical weapons that should kill and/or suppress competitors. With respect to this function it is not surprising that several weapon-like antibiotics have been identified from bacteria living in highly competitive environments like soil.
The great dilemma of anti-infective research is that almost all big pharmaceutical companies have stopped their research and development of new compounds as there is a profit problem in antibiotic R&D. Currently the interest in NPs seems to increase again which is also due to special programmes that support such research by pharmaceutical companies and because of our desperate need for these compounds. Without new anti-infectives we are rapidly approaching a situation prior to World War II where millions of people died of infectious diseases that are still curable at the moment but might not be in 5 - 10 years. As a result of this frightening development, pharmaceutical companies and biotech companies in the United States (US) are encouraged by tax cuts and long lasting patents when they develop new anti-infectives.
Expert knowledge in nematode sampling and characterisation of their symbiotic bacteria from Thailand and Vietnam was used to address the topic of bioprospecting Xenorhabdus and Photorhabdus. These drug producing bacteria live in symbiosis with entomopathogenic nematodes and are used by the nematodes to kill their insect hosts. Although these bacteria are proven producers of natural products, only a few compounds have been isolated and studied in-depth. The isolated natural products were used to build up a unique Xenorhabdus / Photorhabdus-specific compound library which was tested as drug leads for a variety of infectious diseases including anti-protozoal, anti-malaria, anthelminthic, and antibiotic activity. Additionally, insects were used as infection models in order to reduce the need for mammalian testing in preclinical analysis. The complete bioactivity data set for any given compound was collected as basis for future exploitation with pharmaceutical companies. Moreover, the most promising Xenorhabdus / Photorhabdus strains were used in an extensive molecular biology program that would finally result in E. coli strains expressing the biosynthesis pathways responsible for the bioactivity of interest or in Xenorhabdus / Photorhabdus strains over-producing the desired compounds. Finally, the compound library with its connected bioactivity data was made accessible to pharmaceutical companies.
The ultimate goals of the project were therefore the following:
- The isolation of a diverse and large collection of Xenorhabdus / Photorhabdus strains from entomopathogenic nematodes.
- The isolation of new compounds from Xenorhabdus / Photorhabdus strains in order to build up a Xenorhabdus / Photorhabdus-specific compound library that would prove the great potential of Xenorhabdus / Photorhabdus for secondary metabolites.
- Multiple and parallel bioactivity testing against bacteria, fungi, protozoa, nematodes, insects and mammalian cytotoxicity of the compound library.
- Genome mining of the five most promising strains including construction of cosmid / BAC libraries, RVA screening, genome sequencing and heterologous expression in E. Coli.
- In order to attract industrial partners after the duration of the project, all legal issues such as IPR, international agreements and access rights will be settled in advance of licensing the products to companies.
In order to achieve these goals, the project was subdivided into seven work packages (WP1 - WP7). WP1 - WP4 were the core WPs.
WP1 (Strain isolation/characterisation) was the basis for the whole project. A large and diverse strain collection of Xenorhabdus / Photorhabdus strains was obtained from different locations. The work in WP1 was carried out mostly by experts in nematology and microbiology from Thailand and Vietnam.
In WP2 (Chemistry / compound library) all Xenorhabdus / Photorhabdus strains were analysed using analytical methods and new compounds are isolated in a preparative scale from large scale cultivations or synthesized after their structure elucidation to build up a Xenorhabdus / Photorhabdus-specific compound library. This work was carried out by expert natural product chemists in Germany.
These compounds were analyzed in WP3 (Bioactivity testing) against multiple different targets (bacteria, fungi, protozoa, nematodes, insects, cell cultures) in parallel. In order to rapidly identify interesting strains, crude extracts were also tested using the same assays. This work was carried out by biologists well familiar with these standard assays in the UK.
The aim of WP4 (Genome mining) was to identify the biosynthesis gene clusters responsible for the production of the desired compounds allowing their future exploitation and enabling the heterologous production of these compounds in recombinant E. coli. This work was carried out by partners in the UK and Germany.
WP5 (Provision of harmonised set of methodologies) consisted of two sessions. The initial session was required to ensure that all beneficiaries use the same or a comparable methodology regarding especially the bioactivity testing. The consolidation session ensured that the used methods are still comparable but should also involve methods used within WP4 (genome mining).
WP6 (Innovation-related activities) included management of all GAMEXP related intellectual property right issues and coordination of dissemination activities, including the development of the overall dissemination strategy towards the scientific community and the broader public.
WP7 (Management) included the overall responsibility for managing the different work package activities, monitoring timely submission of deliverables and the overall project progress to fulfill the tasks and objectives set out in the work programme, collecting data and preparing interim reports.
Project results:
The following describes selected examples of the results obtains during GAMEXP. As most of the results will be used in final publications that the GAMEXP consortium is currently writing or is sensitive and will be used for additional patents, the presented examples are taken from work already published as part of GAMEXP. A much more detailed description of the GAMEXP results can be found as part of the different deliverables as well as the periodic reports.
In WP1 (Strain isolation and characterisation) more than 1400 soil samples have been collected in Thailand and Vietnam and more than 200 insect-pathogenic Xenorhabdus / Photorhabdus strains have been isolated from these soil samples using an insect baiting technique. All bacterial strains have been analysed using colony morphology as well as recA sequence as morphological marker. Additionally, the bacteria also several of the corresponding entomopathogenic nematode (EPN) partners have been collected and identified using morphological and molecular markers.
In WP2 (Chemistry / compound library) all Xenorhabdus / Photorhabdus strains from Thailand and Vietnam as well as additional strains obtained from public strain collections (DSMZ, ATCC) as well as from other labs working with Xenorhabdus / Photorhabdus, were cultivated on a small scale and the cultures were extracted for subsequent analysis using HPLC coupled to mass spectrometry (HPLC/MS). Here the plan was originally to identify 50 - 100 compounds. However, Xenorhabdus / Photorhabdus were so potent producers of novel chemical diversity, that more than 500 compounds were identified in these strains, most of them unique to Xenorhabdus / Photorhabdus. A MS-MS fragmentation-based database has been set up in order to identify these known compounds but also to identify even more novel compounds that can be analysed in the future.
In order to elucidate the structure of peptides among these compounds based on MS experiments, a new method was developed allowing the determination of the amino acid stereochemistry via a combination of labelling and detailed MS experiments (DOI: 10.1002/chem.201103479) as exemplified for the GAMEXPeptides found in Photorhabdus luminescens. Also as part of the GAMEXP search for novel compounds from Xenorhabdus / Photorhabdus a novel prodrug activation mechanism for non-ribosomal peptides has been identified (DOI: 10.1038/nchembio.688).
A major goal of WP2 was also to build a Xenorhabdus / Photorhabdus specific compound library and at the end this library contains 187 Xenorhabdus / Photorhabdus compounds (these compounds obtained a unique HB# number). This large number of compounds has either been isolated from large scale cultivation of the respective strains or has been synthesized chemically after their structure has been solved by MS and labelling experiments as described above. For peptides, the synthesis is often superior to the isolation of the natural product, especially when the peptides are only produced in very small amounts by the original producer. Among the novel compounds also part of the compound library are the simple phenylethylamides showing an interesting biological activity (DOI: 10.1002/cbic.201100223) just to give one already published example.
The identification of all these compounds based on HPLC/MS also allowed the correlation between phylogeny and compounds present in the individual strains revealing the presence of several highly conserved natural products but also compounds being specific for a subclade of strains or present only in a few species.
Besides HPLC/ESI-MS also MALDI-MS was used for the identification of compounds, which turned out to be especially powerful for very large hydrophobic peptides and highly polar peptides containing several arginine moieties (DOI:10.1021/ac300372p).
In WP3 (Bioactivity testing) the pure compounds were tested against bacteria, fungi, nematodes, protozoa including species causing tropical neglected diseases, insects and insect cells and other eukaryotic cell lines. Here especially antibiotically active compounds have been identified of which some were new or new derivatives of known compound classes (like the xenorhabdins and the xenocoumacins). Even more important, several compounds showed an activity and or selectivity against neglected tropical diseases as tested by the Swiss Tropical and Public Health Institute (Swiss TPH). Among them structurally novel peptide classes but also several low molecular weight compounds. The activity of a few compound classes was in fact so good that further tests are planned in order to evaluate these compounds also in vivo.
When using insect cells as target, microscopic high content analysis was performed as shown for two compounds. Here, differences could be observed even no cytotoxicity could be obtained in some cases.
In WP4 (Genome mining) 15 strains of Xenorhabdus / Photorhabdus were analysed for the presence of biosynthesis gene clusters involved in the production of natural products. Thus, the genomes of several Xenorhabdus / Photorhabdus strains selected based on the production of several different compound classes or the production of conserved and/or bioactive compound classes have been sequenced. Although most genomes were not closed, from the contig data the biosynthesis gene clusters could be identified. Here, we could identify the biosynthesis gene clusters of the most promising bioactive compound class (against tropical neglected diseases) were identified in several different genomes finally resulting in the submission of a patent in order to protect these genes, the corresponding proteins as well as the compounds derived thereof. Additionally, more than 200 other biosynthesis gene clusters have been identified. Several of these have been assigned to a biological activity in the rapid virulence annotation (RVA) approach.
15 biosynthesis gene clusters have been studied in either of the following ways:
- Genes being part of the biosynthesis gene were disrupted or deleted in the original producer, resulting in the production of modified derivatives. This led to the identification of several novel biosynthesis mechanisms, which will also be studied in detail in the future.
- Biosynthesis gene clusters were expressed heterologously in E. coli, resulting in the production of derivatives of the natural products and/or in higher yields.
- The native promoter was exchanged against strong constitutive or inducible promoters in the native producer resulting in the activation of previously silent biosynthesis gene clusters and thus also in the production of the corresponding natural products for the first time (DOI:10.1016/j.jbiotec.2011.10.002).
- The same approach was also very efficient in increasing the yields of already known compounds.
In order to achieve all these goals methods for promoter exchange approach or the hetereologous expression have been developed and/or optimized for Xenorhabdus / Photorhabdus.
For Photorhabdus asymbiotica, which is not only entomopathogenic but can also infect mammals and thus is also an emerging human pathogen, the transcriptome was analysed revealing the presence of several virulence islands activated in human serum or at 37 °C, the temperature of the human body. Here RNA seq methodologies were performed which gave a huge but very robust data set that will also be analyzed in the future. A first result was that also some natural product biosynthesis gene clusters were differentially regulated pointing to the corresponding compounds as virulence factors. Subsequently, compounds were identified, that inhibits the human immune system and thus might play an important part in the pathogenicity of P. asymbiotica.
In order to ensure that all partners use the same methods and know all methods used within GAMEXP, two method manuals (Deliverable D5.1 and D5.2) were written as part of WP5 (Provision of harmonised set of methodologies). D5.1 was dedicated to the isolation of nematodes and bacteria from soil samples and D5.2 was dedicated to all aspects of genome mining used during GAMEXP. Due to the fact that novel tools for genome mining (promoter exchange approach, efficient heterologous expression), D5.2 was updated during GAMEXP.
As part of WP6 (Innovation-related activities) a website for GAMEXP was established (see http://www.GAMEXP.eu(odnośnik otworzy się w nowym oknie) online), allowing also the establishment of the communication structures and its use as data repository for reports and publications. As part of WP6, all intellectual property rights (IPR) issues were solved regarding strains and nematodes or natural products derived thereof. Additionally, SWOT analyses and PUDK analysis were an integral part of WP6.
The management of GAMEXP (WP7) was performed by the company EURICE, specialised in EC-project management. This included the organisation of different project meetings as well as the preparation and submission of project reports.
Potential impact:
-The time has come to close the book of infectious diseases stated by the US General Surgeon William H. Stewart in 1969 has been proven fatally wrong. This statement was given when antibiotic research and discovery had its zenith and new classes of potent drugs entered the market, resulting in the disappearance of many formerly deadly infectious diseases at least in the industrialised nations.
Infectious diseases, however, have made a stunning comeback. Syphilis, gonorrhea, and tuberculosis are all re-emerging with vengeance and the advent of AIDS, hepatitis C, and severe acute respiratory syndrome (SARS) has been a humbling experience. However, the less publicised trend of bacterial resistance may pose an even greater threat. Statistics from WHO hint as to what the future may hold: Vibrio cholerae is 100 % resistant to both tetracycline and chloramphenicol in Tanzania and Rwanda; in some regions, penicillin-resistant Streptococcus pneumoniae makes up 70 % of strains; worldwide, multidrug-resistant Mycobacterium tuberculosis accounts for 1 to 22 % of all new cases. In the US, methicillin-resistant Staphylococcus aureus now accounts for nearly 60 % of hospital-acquired staphylococcal infections, and 20 % of nosocomial infections in US hospitals are reported to be multidrug resistant. Although the numbers are slightly better in Europe, unfortunately Europe is catching up fast.
Even without multidrug resistant strains, nosocomial infections are increasingly important as it has been shown that nearly USD 5 billion are added to US health costs every year as a result of infections that patients get while they are hospitalized for other health problems.
Furthermore, it is reported that that nearly 2 million patients annually acquire infections while being treated for other illness or injury in hospital, and nearly 88 000 die as a direct or indirect result of this infection (4th Decennial Conference on Nosocomial and Health-care associated Infections, Atlanta US). This means, that approximately 1 in 10 hospitalised patients will acquire an infection after admission (Emerging infectious diseases, Vol. 10, No. 4, April 2004). The extra costs arise largely due to the extra days that the patient has to spend in hospital. In general the number of days that a patient has to spend in the hospital varies depending on the type of infection he or she gets: an estimated 1 to 4 days for urinary tract infections, 7 to 8 days for infection at the site of surgery procedure, 7 to 21 days for bloodstream infections and 7 to 30 days for pneumonia. The costs vary from anywhere like USD 600 for urinary tract infections to USD 5000 or more for pneumonia. Prolonged bloodstream infections can top USD 50 000. Other sources reported, that the annual economic costs amount to USD 6,7 billion per year in the US and approximately USD 1,7 billion in the UK indicating that nosocomial infections are in fact in a global problem similarly as infections in general are a global problem.
Even with the adoption of drugs with novel mechanisms of action, we do not have to ask the question if resistance will develop but only when it will occur. This is simply the consequence of the enormous speed at which microorganisms exchange and mutate their genes. It is predictable that the winner in this game will always be the microbe, simply because of its huge numbers, short generation times, and mutation rates.
The best way to combat antimicrobial resistance or at least delay it, is by reducing or, preferably, eliminating the misuse of antibiotics in medicine and agriculture. Moreover, we also have to find new drugs with new mode of actions and therefore new sources of these drugs have to be identified as it is the goal of the current call HEALTH 2.3.4-3 in which this proposal is submitted.
A number of genome sequencing projects within the last five years have revealed several bacterial species that seem to be capable to produce secondary metabolites. Instead the isolation of new species of well-known natural product producers (e.g. actinomycetes) from extreme or not-yet explored environments (e.g. deep sea), one way might be to bioprospect bacterial species that seem to be good NP producers (e.g. Burkholderia, Xenorhabdus, Photorhabdus).
Improving health in general is important in its own right. But is also an important part of the solution to address a number of key challenges facing Europe such as population ageing, security threats or labour shortage. Obviously, infectious diseases play a major role within all these challenges and therefore the question is how bioprospecting projects such as GAMEXP might be involved in their solution?
Possible answers to this can be the following long-term benefits which might arise directly or indirectly from this project:
1. New anti-infective compounds: In an ideal case a compound found within this project is developed into a pharmaceutical product within the next 10 years by a pharmaceutical company to treat one or several diseases.
2. The methods applied within this project (activation of silent biosynthesis gene clusters to increase the chemical diversity) are used for the identification of anti-infective compounds from other organisms.
3. The identification of several biologically active compounds from Xenorhabdus / Photorhabdus will stimulate other research groups or even pharmaceutical companies to explore this huge resource and thereby might lead to the identification of desperately needed cures.
As all current EU problems with infectious diseases are mirrored in countries all over the world, bioprospecting in general will also benefit global health issues, e.g. in Thailand and Vietnam and other developing countries with important tropical diseases (e.g. melioidosis, malaria).
Here it is noteworthy that one of us (Dr Aunchalee Thanwisai who obtained her PhD within GAMEXP) has decided to start her independent career in the field of Xenorhabdus / Photorhabdus in Thailand.
With the aim of expediting the development of new diagnostics, drugs, vaccines and tools for transmission control for effective prevention, treatment and control, the GAMEXP consortium will be established to address several infectious diseases including selected neglected diseases. The proposed work will establish bacteria of the genera Xenorhabdus and Photorhabdus as new source for natural products with potential biological activity. Compounds should be isolated resulting in a library Xenorhabdus / Photorhabdus-specific compounds which are then analyzed in bioactivity tests against a plethora of infectious diseases including several emerging and neglected diseases. This would include assays against multi-resistant Gram-negative and positive bacteria (Vibrio, Pseudomonas, Staphylococcus, Mycobacterium) as well as assay against emerging human bacterial infections (e.g. Burkholderia). Moreover, assays will be conducted against opportunistic pathogenic fungi or yeasts (e.g. Candida involved in AIDS), Acantamoeba as model-protozoa to detect compounds useful to treat trypanosomiasis or leishmaniasis, C. elegans as model for anthelminthic compounds to treat river blindness and filiariasis. Additionally, cytotoxicity against mammalian cell cultures and insects will be determined as model organisms for higher organism.
The compounds with the most promising bioactivity data will be protected. Furthermore, in order to truly establish Xenorhabdus / Photorhabdus as new source of natural products genome mining will be performed that will result also in the finding of the biosynthesis gene clusters responsible for the production of the compounds of interest. This is a prerequisite for biotechnological exploitation of these bacteria.
The unique approach of combining rapid chemical identification, genome mining and massively parallel bioactivity testing in non-animal models will vastly increase the current speed of drug discovery and development. The rigorous patent strategy will also attract partners at an early stage in order to take-over novel bioactives for drug development and deployment.
Regarding general dissemination activities 24 publications in peer-reviewed scientific journals have already been published, three more are already accepted or in press.
Additionally, one patent on the most abundant and novel class of compounds from Xenorhabdus / Photorhabdus showing activity against neglected tropical diseases has been filed. 78 additional dissemination activities (web releases, press releases, newspaper articles) were performed as part of GAMEXP among them 45 talks and presentations given as well as 14 posters presented by GAMEXP members at national and international conferences and meetings.
Project website: http://www.gamexp.eu(odnośnik otworzy się w nowym oknie)
Contact:
Prof. Dr Helge B. Bode,
Goethe Universitat Frankfurt, Germany,
Institut fur Molekulare Biowissenschaften,
Biozentrum/Campus Riedberg (N250, EG),
Max-von-Laue-Str. 9,
60438 Frankfurt am Main
Tel: +49 69 798 29522
Fax: +49 69 798 29527
E-mail: h.bode@bio.uni-frankfurt.de
