Exploitation of Burkholderia bacteria as novel antibiotic producers using a genome mining approach

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

USING NOVEL DISCOVERY TOOLS AND PRODUCER BACTERIA TO IDENTIFY RESISTANCE-BUSTING ANTIBIOTICS

There is an urgent need to discover and develop new antibiotics to treat resistant infections caused by superbugs. Most of the antibiotics we use today are produced by one group of soil bacteria, the Streptomyces. Researchers at Cardiff University discovered that another group of bacteria, known as Burkholderia, produce novel antibiotics that kill superbugs that have become resistant to current antibiotics such as Methicillin Resistant Staphylococcus aureus (MRSA). To enable clinical and commercial development, the chemical structures of antibiotics must be determined to see if they are novel compounds. Via collaboration with experts at the University of Warwick's Department of Chemistry, we have determined complete structures for one Burkholderia antibiotic, enacyloxin IIa, and partially determined the structures of two novel antibiotics, gladiolin and vietnamycin.

Purifying and determining the chemical structures of antibiotics takes time and considerable expertise, therefore researchers are now using a new tool for antibiotic discovery called genome mining. This approach takes the entire genetic code (genome sequence) of an antibiotic producing bacterium and then searches it for genes involved in the production of novel antibiotics. The Wellcome Trust Sanger Institute (WTSI) are pioneers in bacterial genome sequencing and have new technology called Pacific Biosciences sequencing, which is ideally suited to rapid bacterial genome determination in a single sequence run. In collaboration with researchers at WTSI, we sequenced the genome of the gladiolin producing strain and were able to identify the genes involved in production of this antibiotic. Using this extra sequence information, we can now determine the full structure of gladiolin which is needed to take the antibiotic forward into clinical development.

Overall, our preliminary research has shown that Burkholderia are an untapped, promising source of new antibiotics to combat multidrug-resistant superbugs. To determine if Burkholderia can be as successful as the Streptomyces bacteria which produce the majority of the antibiotics we use today, we will use genome mining as a new Burkholderia antibiotic discovery tool, and aim to:

1. Genome sequence 24 antibiotic producing Burkholderia using the Pacific Biosciences sequencing technology. Firstly, to advance characterization of vietnamycin, our anti-MRSA antibiotic, the Burkholderia strain producing this antibiotic will be sequenced. 23 more strains which kill current drug resistant bacteria will then be selected from our large collection. In total, the 24 antibiotic producing Burkholderia genomes will create a unique library of DNA for genome mining and discovery of new antibiotics.

2. Fully identify the structure for gladiolin and vietnamycin, using a combination of chemistry and genetic analysis of their genomes and production pathways.

3. Carry out a unique genome mining survey of Burkholderia antibiotic capacity using the genomes of the 24 strains known to have novel resistance-busting activity. The first two goals of the grant described above will create the genome mining data and also train the researchers on the grant in all the skills necessary to implement this new approach to antibiotic discovery. We will also investigate if "silent pathways" which encode novel antibiotics within the genomes of these Burkholderia can be switched on.

4. The last objective of our research will be to prepare a panel of five novel antibiotics to take forward into preclinical development as drugs to combat resistance. In addition to gladiolin and vietnamycin, we will use the information from genome mining to complete the structure and activity analysis of three further novel Burkholderia antibiotics.

Technical Summary

We discovered that Burkholderia bacteria are an untapped resource of novel antibiotics that can overcome current antimicrobial resistance. Our published data on Burkholderia enacyloxin, a potent anti-Gram negative antibiotic, showed that it is produced by a novel hybrid polyketide synthase (PKS) pathway and is highly exploitable as a member of the successful polyketide antibiotic family. Single Burkholderia strains produce multiple antibiotics and we have now identified a novel macrolide, gladiolin, and another mupirocin-like polyketide, vietnamycin, both active on multidrug resistant Gram positive bacteria. Without genome sequences for these promising antibiotic-producing Burkholderia, the speed at which we can progress both structure and pathway analysis is limited. Therefore we obtained a near complete draft genome of the gladiolin producer strain using long read next generation sequencing from Pacific Biosciences. With few errors and an excellent assembly, the 10 contig draft genome was ideally suited to genome mining, immediately allowing identification of a PKS pathway functionally congruent with gladiolin biosynthesis.

This preliminary data has led us to postulate that "The biotechnological potential of Burkholderia bacteria will be rapidly unlocked by implementing a multidisciplinary genome mining approach to the discovery of novel antibiotics and their biosynthetic pathways." To explore this hypothesis, 24 strains which produce antibiotics overcoming current resistance will be genome sequenced using Pacific Biosciences sequencing and full structure-pathway analysis performed on gladiolin, vietnamycin, and 3 novel antibiotics from the genome mining screen. Methods to switch on cryptic pathways will also be explored to expand the diversity of antibiotics identified by our screens. This genome mining dataset combined with the structure and activity data for a total of antibiotics will establish Burkholderia as a novel antibiotic discovery source.

Planned Impact

Our Burkholderia antibiotic discovery research will have multiple socio-economic impacts on stakeholders as follows:

1. Public health

Antimicrobial resistance (AMR) is now widely acknowledged as being one of the greatest threats to global human health; it also impacts animal health and hence global food security. AMR is World Health Organization priority and UK Government recently published a five year AMR Action Plan (2013-2018; https://www.gov.uk/government/publications/uk-antimicrobial-resistance-strategy-and-action-plan-engagement-exercise). A key action point was to ""facilitate and encourage the development of new drugs" by "encouraging their discovery and development."

Our novel, resistance-busting Burkholderia antibiotics will offer timely solution AMR if they are developed further. It is also likely that other Burkholderia metabolites we discover may have activity on cancer cells or prove useful for the treatment of diseases beyond infection. Our research has broad-reaching potential to save lives and considerably improve the quality of life for individuals who suffer from antibiotic resistant infections. The increased understanding of Burkholderia bacteria as cystic fibrosis pathogens will also impact this group of vulnerable people.

2. Pharmaceutical industry

The UK was once a world leader in natural product antibiotic discovery but due to under investment and the industry shift towards high profit "lifestyle" drugs, the development of new antibiotics by big pharma has largely ceased. The accompanying industry expertise in this area has also been lost. The UK's Chief Medical Officer has recently stated (http://www.bbc.co.uk/news/health-21178718): "There is a broken market model for making new antibiotics, so it's an empty pipeline, so as...[these bugs]... become resistant, there will not be new antibiotics to come."

We are uniquely placed to re-fill this medicinal chemistry pipeline and re-establish the lead the UK once had in natural product antibiotic drug discovery. By filing robust structure-based patents on our Burkholderia antibiotics, we can approach pharma to take on preclinical development of these antibiotics. In addition, the potential to use bioengineering to derive novel antibiotics from the modular Burkholderia polyketide pathways will also bring future benefit to industry.

3. Training of highly-qualified individuals in interdisciplinary drug discovery research

Our research team brings together experts in microbiology, natural product chemistry and genomics, to carry out a state-of-the-art genome mining approach to antibiotic drug discovery. By training in these interdisciplinary skills, the MicroPD and ChemPD will be able to keep pace with cutting edge approaches used in modern antibiotic discovery programs. These skills are vitally needed by both industry and academia.

4. Public education on AMR and the need for new antibiotics.

A key component of the 2013-2018 UK Government Action Plan on AMR was public engagement on the responsible use of antibiotics and awareness that we urgently need to discover and invest in new drugs. The cost of antibiotic development is expensive but the public are used to paying very little for them. Ideologically for example the public are prepared to pay a lot for a new anti-cancer drug and fundraise extensively for these agents. Would they do the same for a new antibiotic? The researchers employed on this grant will explore these dilemmas and philosophical arguments. They will development an AMR/drug development awareness program with schoolchildren in South Wales and the West Midlands, educating them on the need to use antibiotics carefully, develop new drugs and not take continued antibiotic efficacy for granted. Cardiff and Warwick universities have excellent public engagement officers who will assist with these outreach programs.
 
Description We have discovered several novel specialized metabolites of bacteria belonging to the Burkholderia genus, with a variety of potential applications in medicine and agriculture. We identified gladiolin, a novel macrolide antibiotic with promising activity against multi-drug resistant Mycobacterium tuberculosis, as a metabolite of Burkholderia gladioli BCC0238. We also identified cepacin A, originally reported to be produced by Burkholderia diffusa, as a key plant protective metabolite of biopesticidal Burkholderia ambifaria species. Cepacin A was shown to protect germinating crops against Pythium damping-off disease. Several other new metabolites have also been discovered. Manuscripts describing these discoveries have been submitted for publication or are currently in preparation. We have also compiled and published the first comprehensive survey of antibiotics from Gram-negative bacteria, which is not only a valuable resource for us in the context our metabolite discovery program, but the wider community.
In addition, we have developed several important insights into the molecular mechanisms responsible for assembly of specialized metabolites in Burkholderia species. We have identified the biosynthetic gene cluster for cepacin A, opening the path to understand the unusual biosynthetic pathway for this metabolite. We have also located the gladiolin biosynthetic gene cluster, enabling us to investigate several intriguing facets of the "trans-AT" polyketide synthase responsible for gladiolin assembly. This has already resulted in the discovery of a new mechanism for subunit interaction in polyketide synthases, which is fundamentally different from those reported previously. Manuscripts reporting several other interesting aspects of gladiolin biosynthesis are in preparation and will be submitted for publication soon. The biosynthetic gene clusters for several other novel Burkholderia metabolites have also been identified, enabling novel aspects of their biosynthesis to the investigated. The results of these studies will be published in due course.
Exploitation Route Our findings have the potential to be developed by pharmaceutical, agrochemical and biotechnology companies. Gladiolin has promising activity against multi-drug resistant tuberculosis and offers the potential to be developed into a new therapy. A patent has been filed and granted for this, but it has not been maintained by the Universities involved, due to the economic challenges associated with commercializing new antibiotics for the treatment of tuberculosis.
Through additional BBSRC funding (BB/S007652/1), we are investigating the development of Burkholderia ambifaria as a safe and efficacious biopesticide. We are also investigating the molecular mechanism of cepacin A biosynthesis, as a prerequisite to analogue creation via biosynthetic pathway engineering. This will enable a better understanding of the structure-activity relationship of this antibiotic, facilitating its development as a pesticide.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC Committee D responsive mode
Amount £742,036 (GBP)
Funding ID BB/R01218/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description BBSRC Future Leader Fellowship
Amount £300,000 (GBP)
Funding ID BB/R012121/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 01/2021
 
Description BBSRC NPIF Industrial CASE training grant
Amount £98,212 (GBP)
Funding ID BB/R505845/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description Establishing the Efficacy, Safety and Persistence of biopesticides based on naturally occurring beneficial bacteria
Amount £860,000 (GBP)
Funding ID BB/S008020/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 03/2022
 
Description Cardiff 
Organisation Cardiff University
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team focused on the discovery and biosynthesis of new antibiotics and other bioactive natural products from the Burkholderia cepacia complex.
Collaborator Contribution Our collaborators screened their collection of Burkholderia cepacia complex bacteria for antimicrobial activity and coordinated genome sequencing and comparative analysis of the collection.
Impact Three papers have already been published. Several more are in preparation.
Start Year 2011
 
Description EPFL 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution Supplied novel antibiotics isolated from Iranian Actinobacteria and Burkholderia gladioli for biological testing
Collaborator Contribution Tested compounds for activity against M. tuberculosis, including activity against several MDR clinical isolates
Impact Multidisciplinary collaboration involving Natural Products Chemistry and Microbiology. One paper published in J. Am. Chem. Soc. in 2017. Other outputs are in preparation for publication.
Start Year 2017
 
Description Richard Ebright 
Organisation Rutgers University
Country United States 
Sector Academic/University 
PI Contribution We have supplied samples of the antibiotic gladiolin to our collaborators
Collaborator Contribution Our partners are attempting to determine the structure of the antibiotic-RNA polymerase complex
Impact None yet
Start Year 2017
 
Title ANTIMICROBIAL AGENTS 
Description The invention providesnovel macrolide compounds of formula (I) and their pharmaceutically acceptable salts, metabolites, isomers (e.g. stereoisomers), and prodrugs. In formula (I), R1 is hydrogen or an ester-forming group; R2 is methyl or oxo; R3 is methyl or hydroxyl; R4 to R8 are each independently hydrogen or methyl; and R9 to R14 are each independently hydroxyl or oxo. Methods for preparing such compounds, pharmaceutical compositions comprising them, and their use as antimicrobial agents also form part of the invention. The invention further provides a new strain of bacteria, Burkholderia gladioli strain LMG-P 26202, which is capable of producing the macrolide compounds. 
IP Reference WO2015145152 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact This discovery was covered by an article in the economist when we published it in the academic literature in 2017.
 
Description CREST Gold Award 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The researcher working on the project hosted two sixth form students working towards a CREST Gold Award for a one week project in the lab.
Year(s) Of Engagement Activity 2016